Pooley, who won silver in the Olympic time trial in Beijing and has been one of the form racers this season, clocked a winning time of 32 minutes 48 seconds to beat Germany's Judith Arndt into second place by 15secs in Australia. Linda Villumsen finished in third, 15.8sec behind the Briton, to take the bronze medal for New Zealand.
A climbing specialist who only took up the sport in 2005, Pooley's bid for gold almost came unstuck when International Cycling Union (UCI) officials said her time trial bike did not comply with regulations.
Pooley therefore had to use her training bike and, after making a few last-minute adjustments, got on with the job in hand.
The Cambridge graduate, however, stayed calm and put her climbing skills to good use on the hilly 22.9-kilometre circuit although she was equally impressive on the descents and on the long flat 12km run to the finish, areas of a long time trial in which she has previously struggled.
Two years after she finished second behind Kristin Armstrong in Beijing, Pooley took full advantage of the American's retirement to stamp her authority on the race.
"The Olympic Games is pretty special but, in a way, that was easier for me because I had no expectations and no pressure," said Pooley afterwards. "This time it was different. I trained specifically for this, doing a lot of hill training and intervals on my time trial bike. Now I get to wear the world champion's jersey with the stripes for a whole year."
"Luckily I have two bikes, one to train on and one to race on, and at the last moment, I was racing on the training bike and maybe I haven't raced on it for a while. It hasn't been checked and they [the UCI] were very strict. But at the end of the day, it's more about the legs than the bike."
"It's fantastic, I'm really happy. I guess it all went well and I quite like the fact that this year no radios were allowed so no one was getting intermediate time checks which meant all you could do is ride your fastest and the fastest person wins. I prefer not to think about the other competitors, just do the best I can and see what happens at the end."
Meanwhile Arndt, one of the most successful women racers of her generation but who now has three silver and one bronze from the championships' time trial, was visibly upset.
"It's always nice to win a medal at the world championships, but just one time I would like to win the gold," said the 34 year-old German, who won the world road race title in 2004 when she also took Olympic road race silver in Athens.
Villumsen picked up her second consecutive bronze and achieved the rare accolade of becoming the first woman to win world championship medals for two different countries. Last year Villumsen won bronze for her native Denmark, who will host the competition in 2011, but said she is now proud to be a Kiwi.
"I feel New Zealand is my home now and I'm proud to represent the country. But hopefully in the next few years I will win the gold," said Villumsen.
Jeannie Longo, the 51 year-old Frenchwoman, who has multiple world and Olympic titles and a total of 57 national titles, finished just off the podium place in fifth, 43secs behind Pooley. Having beat a host of rivals who were not even born when she competed at her first Olympics in 1984, Longo showed that she has every right to be considered for selection at the London Games in 2012.
"I'm satisfied with my performance despite just missing the podium,” said Longo, who refused to rule out racing at the London Games.
"I'm frightened of being too old. But there's only one year to go and preparations will begin."
