Wayne is a native of Fort Lauderdale, but has also lived in Houston, Washington DC, New York and graduated from Kaiser High School in Honolulu. He graduated from the University of Florida with a B.S. in broadcast journalism in 1993.

Wayne worked in corporate communications for Edelman Public Relations Worldwide and as a press secretary for democratic Maine State Senator Sean Faircloth's bid for the U.S. Senate. In 1992 Wayne co-founded Sons & Daughters of America (SDA), a gay and lesbian public awareness campaign based in Fort Lauderdale.

He is a gay rights advocate in the United States. He is a former spokesman for the Human Rights Campaign, the nation's largest gay and lesbian advocacy group. He is an author, activist, columnist and public speaker. Wayne has also spoken at many leading universities, including Vanderbilt, Harvard Law School, John Hopkins, and the University of Florida.

Wayne, a gay man, was never personally involved in the ex-gay movement, but says he has interviewed hundreds of former and current ex-gays. In 2000, he photographed ex-gay activist John Paulk in a Washington D.C. gay bar called Mr. P's. Paulk said he was simply there to use the washroom, but Besen and other witnesses allege he was drinking and flirting for over twenty minutes. Besen went public with the story, and wrote about it in his book Anything But Straight: Unmasking the Scandals and Lies Behind the Ex-Gay Myth.

The book was nominated for two Lambda Literary Awards in 2003.

Besen's photograph of Paulk (and subsequent release of the story) was instrumental in the ultimate removal of Paulk as Chair of Exodus International, a major organization in the "Ex-gay movement". As noted by The Washington Post in October 2002: "[John Paulk] had been the most famous success story of the Christian ex- gay movement, which seeks to persuade gay men and lesbians to accept Jesus and renounce homosexuality. He had appeared on "60 Minutes," "Oprah" and the cover of Newsweek."

In June 2006, Besen founded Truth Wins Out, a non-profit organization to counter ex-gay ministries' myth and right wing propaganda. He also launched www.RespectMyResearch.org, to document right wing distortions of science, particularly by Focus on the Family.

His hobbies include playing and watching basketball, traveling, reading and writing.