Part religious education centre, part adventure playground, Ark Encounter is based on the biblical tale in which God instructs Noah to build an ark to save his family and a pair of each of the world's animals from perishing in a great flood.
The state of Kentucky has experienced severe flooding coinciding with the opening of the attraction.
The ark-shaped structure is purported to be the world's largest timber-framed building and was designed by the Indiana firm Troyer Group.
Constructed according to biblical dimensions by Amish carpenters, it spans over 155 metres in length, is 26 metres wide and 16 metres tall.
The park is positioned in Williamstown, a city just south of Cincinnati, making it within a day's drive for two thirds of the US population. It is run by the Christian fundamentalist organisation Answers in Genesis as a sister attraction to the nearby Creation Museum.
"In a world that is becoming increasingly secularised and biased, it's time for Christians to do something of this size and this magnitude," said Answers in Genesis chief executive Ken Ham.
The attraction is designed to accommodate 10,000 visitors a day, but with high demand expected in its first weeks, it will have extended opening hours for the next 40 days and nights – the duration of rainfall during the biblical flood.
Inside the ark building, three timber-lined floors host exhibits exploring how Noah, his family and animals might have lived on board the ark. Enclosures feature life-sized animal models, including dinosaurs.
A gift shop and a 1,500-seat-restaurant named Emzara's Kitchen after Noah's wife are also contained within the building.
The 800-acre site also includes the Ararat Ridge Zoo, which is populated by Tibetan yaks, kangaroos and ostriches, as well as a petting zoo offering camel and donkey rides.
Finishing touches are also being added a nine-mile-long zipline course, which is set to transport visitors at speeds of up to 50 miles per hour through the grounds.
These will conclude the $100 million-works on the first phase of the development, which was originally unveiled in late 2015.
Future phases are to include the construction of a rooftop restaurant, a replica of a first-century Middle Eastern village, "the Tower of Babel" and a walk-through aviary.