A new kind of packaging film was prepared using tilapia collagen as substrate. The influence of collagen, glutaraldehyde, sodium alginate, and glycerol on the mechanical properties of the edible film was investigated. Response surface analysis test with three factors and three levels was design according to Box-Benhnken center composite design principle. Adding amounts of collagen, glutaraldehyde, sodium alginate, and glycerol was used as test factors and tensile strength of the packaging film as response index. The interaction between factors was discussed, and optimum technical formula for tilapia collagen packaging film was obtained.