The replica was built by Christians who said they believe the biblical story was an historical event.
Critics have said the attraction should not have received tax incentives as it contradicts science education.
The ark is based on a biblical story of a man who received an apocalyptic warning from God about a massive flood.
Answers in Genesis, the ministry that built the ark, said the ship's dimensions are based on those described in the Bible.
The ark is also 85ft (26m) wide and 51ft (15m) high, according to the group.
The ark, which is about 40 miles (64km) south of Cincinnati, Ohio, in Williamstown, features replicas of animals in cages, displays of Noah's family as well as exhibits on the group's theories on the world's existence.
The group believes that God created everything, including dinosaurs, about 6,000 years ago, despite scientists have said the dinosaurs became extinct about 65m years before man appeared.
Ken Ham, president of Answers in Genesis, said the total cost of the ark is more than $100m (£77m).
The organization estimates that the theme park, which debuted to the public on Thursday, will draw about two million visitors in its first year.