What started as a pilot in 2013 has grown into a database "without any public debate or oversight", said the American Civil Liberties Union.
It amounted to "runaway surveillance", director of technology Nicole Ozer tweeted.
The FBI said it was developing "best practices" for iris image capture.
The project was launched in September 2013 and has seen the FBI collaborate with agencies in Texas, Missouri and California.
The iris data, taken from people who have been arrested, can be scanned in a fraction of a second.
The scan takes a detailed image of the ridges in the coloured part of the eye, which are as detailed and distinctive as a fingerprint.
An average of 189 iris scans were collected every day in California at the start of 2016, according to documents obtained by The Verge.
The programme was started to "evaluate technology, address key challenges and develop a system capable of performing iris image recognition services", according to the FBI's website.
Such technology is necessary in order to easily track criminals and quickly catch repeat offenders and suspects who try to hide their identities, the FBI argued.
The project falls under its $1bn (£750m) next-generation identification system that aims to expand the bureau's old fingerprinting database to other identifiers such as facial recognition and palm prints.