Lane was born Morton Donald Isaacson at the Manhattan "Flower Hospital" in New York City to a Jewish father (Jacob) and a Catholic mother (Dolly), who later converted to Judaism. Jacob "Jack" Isaacson was a sergeant in the New York City Police Department (NYPD) and Dolly was a homemaker. He was raised in The Bronx, where he attended DeWitt Clinton High School and was classmates with Judd Hirsch and Garry Marshall. By his mid-teens, due to his mother's influential personality, Isaacson had decided he wanted to be in show business.

In 1965 the Irish comedian Dave Allen presented a talk show on Sydney television for TCN-9. He was eventually sacked and Nine producer John Collins looked for replacement hosts to fill in for the rest of the season. After a series of temporary hosts, Collins found Lane working in The Dunes Nightclub in Hawaii. While in the United States, Collins asked Las Vegas performer Wayne Newton if there was anybody he should consider as a replacement; Newton's answer was "Don Lane".  Lane was offered the job after filming and sending in an audition tape for the TCN-9 executives. He was given the host's chair for six weeks. He planned to base his version of the show on Johnny Carson's Tonight Show. Lane's run was variously referred to as The Tonight Show, Tonight with Don Lane and Sydney Tonight. Within a month, Nine settled on Lane as permanent host. After four weeks, his original six-week contract was extended to forty weeks.  Tonight with Don Lane featured comedy sketches, interviews with visiting entertainers and musical numbers often performed by Lane with the Tonight Show Orchestra. The announcer for the show was Mike Walsh, who later went on to host his own successful variety show.  Due to technical restrictions, Lane's tonight show was only broadcast throughout New South Wales, just as In Melbourne Tonight(IMT) was restricted to Victoria. Work on a coaxial cable linking Melbourne with Sydney had begun in June 1959 and was completed on 5 February 1963. On 7 July 1965, Lane appeared on a then-innovative live split-screen link with Graham Kennedy via the cable. Lane and Kennedy sang the song "Seventy-Six Trombones" as a duet. They took full advantage of the new technology and the duet included the performers throwing jugs of water and Kennedy holding up a sign saying "Go Home Yank", to which Lane laughed. Lane also performed another duet with Kennedy at the Logies ceremony in 1967.  In March 1968, Lane was charged with importing marijuana into Australia. He was arrested at Sydney Airport and remanded in custody. He strenuously protested his innocence, claiming that the drugs were planted into his jacket pocket by a former business associate who wanted revenge. He was found not guilty on all charges, being defended by barrister Marcus Einfeld.

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