At precisely 4:10 a.m. - the time of the shooting - the mourners rang a large bell 50 times to mark the number of bullets fired at the Bell and two of his friends, who were seriously injured.
After laying wreaths and flowers, the group marched half a mile to a church, where Sharpton held a prayer service.
"The idea is to commemorate the second anniversary and to continue to push for a federal case," Sharpton said later.
At a non-jury trial in April, a judge acquitted three police officers of state charges that included manslaughter, assault and reckless endangerment.
The officers - undercover detectives investigating reports of prostitution at the club - said they opened fire because they thought one of the men was reaching for a gun. No weapon was found.
The shooting and subsequent acquittals of the officers ignited protests and raised questions about whether the NYPD was too quick to use excessive force against minorities.
Last week, Bell's family and their lawyers met privately with federal prosecutors to discuss a possible civil rights case against the shooters.
"They've assured them they were doing a full investigation," Sharpton said Tuesday. "We don't know what the outcome will be but ... the family is still demanding justice in this matter."
U.S. Attorney Benton Campbell has refused to comment on the private meeting.
"Sean Bell's civil rights were violated - the right to go home unarmed and not be killed by police," Sharpton said.
Among others in attendance Tuesday were Bell's parents and Joseph Guzman, one of the two men injured in the shooting.
"It seemed like it just happened all over again just being here," Bell's father, William Bell, told NY1 television. "The feeling hasn't changed. It still hurts."