The officers, acting on tip-off from US counterparts, seized the 649-kilogram (1,431-pound) haul from a container from Ecuador as it was being transported by truck to an industrial area in the remote New Territories on Wednesday.
Authorities intercepted the truck, arresting three local men including the driver.
"This is the biggest ever cocaine seizure made in Hong Kong," Customs Drug Investigation Bureau head John Lee told reporters. He said officers found 22 bags with a total of 541 slabs, each with around 1.2 kilograms of cocaine.
Lee said he believed most of the cocaine was to be transported to Southeast Asian countries, but denied the southern Chinese city has become a transit route for drugs smuggling.
The seizure was the second in a week. On Tuesday, a 63-year-old man travelling from Brazil was arrested for allegedly smuggling almost two kilograms of cocaine in his specially adapted underpants and shoes.
Hong Kong's previous biggest cocaine bust was in September last year when 567 kilograms of the drug was seized and eight people - from the United States, Columbia and Mexico - were arrested.
Ecuador, situated between the world's two biggest cocaine producers, Peru and Colombia, is often used as a transit point for traffickers seeking to transport drugs into the region.