Thousands of protesters rallied in and around Lung Wo Road, an important east-west artery near the offices of embattled Hong Kong Chief Executive Carrie Lam, as hundreds of riot police warned them to stop advancing.
Some protesters erected barricades to block traffic in the heart of the Asian financial center, with many defying police calls to retreat, in scenes reminiscent of pro-democracy protests that rocked the city in late 2014.
The government advised staff to avoid driving to government buildings because roads were blocked.
Lam has defiantly vowed to press ahead with the controversial legislation despite deep concerns across the Asian financial hub that triggered on Sunday its biggest political demonstration since its handover from British to Chinese rule in 1997.
Demonstrators from across a wide spectrum of Hong Kong society began joining the overnight protesters earlier on Wednesday as businesses across the city prepared to go on strike.
The bill, which has generated unusually broad opposition at home and abroad, is due for a second round of debate on Wednesday in Hong Kong's 70-seat Legislative Council, although it was not immediately clear if that would go ahead as planned.
The legislature is controlled by a pro-Beijing majority.
Lam has sought to soothe public concerns and said her administration was creating additional amendments to the bill, including safeguarding human rights.
In a rare move, prominent business leaders warned that pushing through the extradition law could undermine investor confidence in Hong Kong and erode its competitive advantages.
Sunday's protest, which organizers said saw more than a million people take to the streets, in addition to a snowballing backlash against the extradition bill could raise questions about Lam's ability to govern effectively.
That protest rally plunged Hong Kong into political crisis, just as months of pro-democracy "Occupy" demonstrations did in 2014, heaping pressure on Lam's administration and her official backers in Beijing.
A spokesman for bourse operator Hong Kong Exchanges and Clearing (HKEX) said a cocktail reception on Wednesday evening to celebrate 19 years of being listed, at which Lam is guest of honor, would go ahead.
The protesters, mostly young people, wore makeshift protective gear such as masks and goggles as they dragged steel barriers on to roads, wreaking commuter havoc in the morning rush hour.
The demonstrators rallied just a stone's throw from the heart of the financial center where glittering skyscrapers house the offices of some of the world's biggest companies, including HSBC.
HSBC and Standard Chartered, in addition to the Big Four accounting firms, had all agreed to flexible work arrangements for staff on Wednesday, Hong Kong media reported.
Strikes and transport go-slows were also announced for Wednesday as businesses, students, bus drivers, social workers, teachers and other groups all vowed to protest in a last-ditch effort to block the bill.
The Catholic Diocese of Hong Kong called on the government not to pass the bill "hurriedly" and urged all Christians to pray for the former British colony. Lam, who warned against "radical action" at the latest protest, is a Catholic.
Britain handed Hong Kong back to China 22 years ago under a "one-country, two-systems" formula, with guarantees that its autonomy and freedoms, including an independent justice system, would be protected.
However, many accuse China of extensive meddling since then, including obstruction of democratic reforms, interference with local elections and of being behind the disappearance of five Hong Kong-based booksellers, starting in 2015, who specialized in works critical of Chinese leaders.
Beijing rejects those accusations and official Chinese media said this week "foreign forces" were trying to damage China by creating chaos over the extradition bill.
Human rights groups have repeatedly cited the alleged use of torture, arbitrary detentions, forced confessions and problems accessing lawyers in China, where courts are controlled by the Communist Party, as reasons why the Hong Kong bill should not proceed.China denies accusations that it tramples on human rights.