The Islands project is one of the biggest zoo developments in Europe.
O’Rourke will create a series of bridges and a river ride touring vistors around animals and plants from the southeast Asian islands.
The Islands is set to open to the public in 2015.
Simon Mann, Chester Zoo’s Development Director, said: “This marks a new stage in the development of Chester Zoo and it will be unlike anything experienced before in a UK zoo.
“Islands is a complex project that includes the creation of landscaping themed to each island, an exotic boat journey and world-class animal exhibits which marry the exact needs of our animals with the best experience for visitors.
“We’re bringing these islands to life, right here in the heart of leafy Cheshire, and making the opportunity to visit these far-flung places a reality for many people.
“Finding a company who can understand our ethos and work confidently with us through the build process was vital.”
Laing O’Rourke will also be working with the zoo to host open days for local residents to find out more about the build.
Tom Higgins, who leads Laing O’Rourke’s northern business, said: “Laing O’Rourke is delighted to be working with Chester Zoo to deliver such an exciting project for the North West.
“We’ll apply the latest engineering solutions to the project, including digital engineering and Design for Manufacture and Assembly; techniques that will offer best value and allow for a more sustainable and safe delivery.
“We also share with Chester Zoo a strong focus on education, and we look forward to working with local schools and education providers as we deliver The Island project.”
The Islands will also include a major new Indonesian jungle house, which will be the largest indoor zoo exhibit in the UK and home to Sumatran orangutans, Sulawesi macaques and a new species for the zoo, the sunda gavial crocodile.
It is expected that it will take zoo visitors two hours to journey their way around Islands.