The Swiss has not had the ideal preparation for the Tour, as he fractured his collarbone in April in the Tour of Flanders and his RadioShack team has been hampered by internal conflict and the withdrawal of team leader Andy Schleck through injury.
But Olympic and four-times world champion Cancellara clocked seven minutes and thirteen seconds in Liege, at an impressive average speed of 53.2kph, to beat Tour de France favourite Bradley Wiggins of Britain by seven seconds.
"It's pretty high on my list of yellow jerseys ... yes, it's on the top for sure," said the 31-year-old Cancellara, who won his first major victory on an almost identical course in the Belgian city eight years ago and has since collected 22 yellow jerseys.
"It's phenomenal to win eight years later. So much happened since 2004. I watched the video only two days ago. I was 23. I think the junior I was inspired me today."
French time-trial champion Sylvain Chavanel was third in the same time as Wiggins. Defending Tour champion Cadel Evans finished a solid 13th, 17 seconds adrift.
Like Wiggins, Cancellara has a crucial year ahead of him.
But he refused to talk about the Olympic Games, which begin in London next month.
Cancellara said: "The important thing is tomorrow, and then we'll see every other day. Who knows where I'll be in one month? Nobody could expect this bottle to be on the road when I crashed on April 1. I'm taking every day as the last one."
Another special motivation for the Swiss is the forthcoming birth of his second child.
"This is for my wife, Stefanie," he said. "She gave me a lot of strength, as does the baby who's coming."
The Tour opener left Wiggins with mixed feelings -- he was a disappointing 11th at half-course but finished fast to salvage a podium spot.
"I was really calm and relaxed and kept putting myself back to reality with my iPod to isolate myself from all this madness," Wiggins said, referring to the large, enthusiastic crowds lining the course in the streets of Liege.
"It's nice to get started on the road. I just tried to focus on myself and not take the others into account."
It was the third time this season the Team Sky leader finished a close second in a major stage race prologue after Paris-Nice and the Dauphine.
Since he ended up winning both races, it could be seen as a good omen for the man aiming to become the first Briton to win cycling's showcase event.
Tejay van Garderen, Evans's young team-mate, took fourth place and secured the best young rider white jersey.
Other favourites did not fare so well, however.
Time-trial world champion Tony Martin of Germany punctured as he was leading the way. He was forced to change bikes but still managed to finish only 23 seconds off the pace.
And Slovakia's Peter Sagan narrowly avoided crashing when his foot skidded off the pedal. He finished 52nd.
On a broader scale, Cancellara's victory was a relief for his RadioShack team, which has been rocked by internal trouble in the past few months.
"It takes a lot of pressure off the team. As for myself, I'm looking forward not back," said Cancellara.
There have been reports that brothers Andy and Frank Schleck were at odds with team manager Johan Bruyneel and might leave the team at the end of the season.
Bruyneel has stayed away from the Tour after being cited in an investigation by the US Anti Doping Agency (USADA) into seven-times Tour champion Lance Armstrong.
Sunday's 198-kms first stage to Seraing finishes at the top of a short but steep hill and could favour riders such as Sagan or local hero Philippe Gilbert, who won the first stage a year ago.