The 31-year-old London-born rider on Tuesday announced her intention to retire after Sunday's road race in which she will support Lizzie Armitstead.
Pooley had individual ambitions first in the 29.6-kilometre time-trial, which began and finished at Glasgow Green, and led at every time check before finishing in 42 minutes 31.49 seconds.
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However, Linda Villumsen of New Zealand dug deep in the final stages to triumph in 42mins 25.46secs.
Katrin Garfoot of Australia was third in 43:13.91.
Pooley was the penultimate rider to roll down the start ramp and held a slender advantage of less than two seconds over Villumsen at the first two time checks on slick roads.
The 2008 Olympic silver medallist and 2010 world champion enhanced her advantage to more than eight seconds with six kilometres to go, with the gold between her and Villumsen.
Pooley passed Australian Shara Gillow, who began one minute ahead of her, and dashed for the line, but could only watch as Villumsen powered through to steal gold.
Scotland's Katie Archibald was firmly in contention to join the pair on the podium, in third at the first two time checks, but she slipped down the order in the closing kilometres.
The Milngavie rider finished fifth in 43:30.01.
Wales' Elinor Barker, a former world junior champion in the event, was seventh in 43:56.44, while Joanna Rowsell, the individual pursuit champion on the track, clocked 44:45.87 to place 13th.