The prisoners are said to be members of the Libyan Islamic Fighting Group (LIFG) and had been serving prison terms ranging from 10 years to life imprisonment.
Most had already spent between six and eight years behind bars in the capital's main prison, Abu Salim.
The group, which first became public in 1995, aims to oust Gaddafi who has led the country since overthrowing the monarchy in 1969.
The government-funded Gaddafi International Foundation for Charity Associations is said to have held talks for months with the organisation's leaders.
The charity said that those released represent a third of the group's members held in Libyan prisons.
Opposition
According to an audio tape released on the internet last November and attributed to Ayman al-Zawahiri, an al-Qaeda leader, the group had affiliated itself with al-Qaeda.
One of those released said: "Our religion and our society do not accept violence."
Saleh Abdul-Salam, director of the charity, said: "All the released have denounced violence.
"The foundation will help them in finding work and integrating better into society."
Talks are ongoing for the release of an estimated further 180 detainees.