The previous record of securities turnover was $211bn on 3 October 2007.
"The Stock Connect was one of the catalysts for Wednesday’s record high securities market turnover," said C K Chow, chairman of the exchange.
"The programme has given us another source of market liquidity that was enhanced by the mainland authorities' recent clarification of policies regarding institutional investor participation in the Stock Connect," Chow said.
Continuing with its capital market reforms drive, the Chinese Securities Regulatory Commission recently allowed domestic mutual funds to invest in Hong Kong shares using the Stock Connect programme.
The southbound Stock Connect, which allows investors in China to invest into Hong Kong markets, had a turnover of RMB16.8bn ($2.7bn), surpassing the previous high of RMB4.8bn on 2 April.
For the first time, the RMB10.5bn daily quota for southbound trading was also reached.
The northbound trading link, allowing Hong Kong investors to buy in China domestic market had a turnover of RMB13.2bn, surpassing the previous high of RMB12.1bn on 17 November.
The Hong Kong exchange will continue to ensure that the market operations remain smooth as turnover rises, Charles Li, chief executive of the Hong Kong Exchange said.
“When turnover grows, we always take appropriate risk management measures as required to ensure our market integrity,” Li added.
A survey by Hong Kong Fund Investment Association conducted in December showed the programme did not meet early market expectations due to investors’ concerns over technical, legal and tax issues.
The same survey also found that more than half of the respondents were positive on the potential of the Stock Connect this year.