Berntsen grew up in Smithtown, Long Island, a suburb of New York City. When Berntsen was seventeen, he tried to join the United States Army but his parents would not sign the enlistment contract. He waited until his eighteenth birthday he decided to enlist in the Air Force. Berntsen served for four years as a Crash Firefighter in the Air Force, including tours in Alaska and Korea. During his time in the United States Air Force he became obsessed with educating himself, which he did by reading biographies and enrolling in the Air Force's self-study program, acing several courses. Berntsen also spent much of his time in the gym or skydiving. After Berntsen's time as a firefighter he became a full-time college student where he majored in Political Science and minored in Russian Studies. He attended the U.S. Marine Corps Platoon Leader Class for two summers. However, before he was commissioned in the Marine Corps, he was recruited into the CIA.

In August 1998 Berntsen led the response team to the U.S. Embassy in Dar es Salaam. The subsequent investigation led to the capture of some 21 individuals involved with the planning and execution of the attacks.

Berntsen went to Afghanistan in March 2000 as a part of a CIA team that planned on capturing a senior Al Qaeda leader. Berntsen writes that though leaders at the CIA, particularly Cofer Black and Hank Crumpton "had shown a willingness to plan and execute risky missions," top US government leadership including President Bill Clinton and CIA Director George Tenet were unprepared to take the increased risks. It is recognized that the environment for covert CIA operations in areas of Central Asia significantly shifted after the September 11 attacks in 2001 on New York City and Washington, D.C. The legitimacy for conducting such operations received widespread recognition and the actions carried out by CIA personnel after the attacks allowed for greater efficacy in targeting Al Qaeda personnel planning and conducting operations against US interests. Furthermore, Berntsen's mission was also undermined by a CIA station chief in a neighboring country, dubbed Lawrence, who contacted CIA Headquarters calling for the mission to be called off. The mission was never executed and the CIA's indecision would lead the Northern Alliance's leader Ahmad Shah Massoud to conclude that the US was "not serious" about fighting the Taliban.

In December 2001, Berntsen served as the Commander of all CIA forces in Eastern Afghanistan and led the agency’s Jawbreaker team in Tora Bora. With a new President and the United States rattled by 9/11, Berntsen returned to Afghanistan with significantly different instructions. On September 19, 2001, Cofer Black, head of the CIA’s Counterterrorist Center told Berntsen, "You have one mission. Go find the al-Qaeda and kill them. We’re going to eliminate them. Get bin Laden, find him. I want his head in a box. I want to take it down and show the president."

In his 2005 book, Jawbreaker, he alleges that Osama bin Laden could have been captured at Tora Bora if the US military (specifically United States Central Command) had devoted more resources to the operation. This claim gained substantial traction due to a Senate Report on the circumstances of bin Laden's escape. According to both Berntsen's account and the Senate Committee's report, "Bin Laden and bodyguards walked unmolested out of Tora Bora and disappeared into Pakistan's unregulated tribal area." Berntsen insists this would have been stopped by a US military presence on the Afghan-Pak border, instead of a reliance on corrupt local warlords.

In July 2005, Berntsen took the CIA to court alleging that they were violating his First Amendment rights and the Administrative Procedure Act by taking longer than the allowed 30 days to approve his Jawbreaker manuscript for publication. This delay caused him to miss the June 17 deadline for handing over the manuscript to his publisher and caused the book to miss its original October publication date.

From June 2007 to June 2008, Berntsen returned to Afghanistan as a civilian advisor on Improvised Explosive Device networks for Regional Command East (RC-East), within NATO-led International Security Assistance Force.

Gary released his first piece of fiction in August 2008. The book is titled The Walk-In and tells the story of an American CIA case officer dealing with an Iranian defector from the Quds Force. The defector claims that a catastrophic attack is imminent and the American case officer must decide what to believe.

In November 2008, Berntsen published Human Intelligence, Counterterrorism, and National Leadership: A Practical Guide. This book was written to serve as a manual for the incoming president and White House staff and includes highly specific recommendations and policy prescriptions for human intelligence and counterterrorism operations.

Berntsen continues to influence US policy by expressing his expert opinions and analysis to the news media and several government entities including police departments and federal agencies.

Main Article: United States Senate election in New York, 2010

On May 23, 2010, Berntsen officially announced his candidacy for the New York Senate Seat currently held by Chuck Schumer. Berntsen's political campaign believes his national stature, his current ability to effect public policy, and his experience as a clandestine CIA operative make him a strong candidate for elected office. On June 1, 2010, with considerable support from the New York Tea Party movement, Berntsen is the Republican designee for US Senate against incumbent US Senator Chuck Schumer from New York and faces a Republican primary against Jay Townsend who received the Conservative party designation. After the GOP designation, personal endorsements from Republican leaders in New York began accumulating for the Berntsen campaign, including Representative Peter King. In addition to the Republican nomination, Carl Paladino declared Berntsen his nominee for Senate on his "Taxpayers Party" line.

Jay Townsend defeated Berntsen in the September 14, 2010 primary, 55 percent to 45 percent.