The aim of this paper was to optimize complex biological preservatives consisting of citric acid, thymol and sodium alginate using three-variable, three-level Box-Behnken design to prolong the shelf life of Takifugu rubripes. A quadratic polynomial regression model was established with total plate counts as the response value. The preservation effects of complex biological preservatives were verified by measuring pH, drip loss, TVB-N, TBA, total plate counts and Pseudomonas sp.. The result showed that the optimal ratio of the complex biological preservatives at ice temperature is as follows: 0.68% citric acid, 1.45% thymol and 1.94% sodium alginate. In that condition, the lowest total plate count was 5.50 lg (CFU/g) on the 30th day. What’s more, citric acid and thymol, citric acid and sodium alginate, as well as thymol and sodium alginate had a significant interactive effect. The optimal complex biological preservatives could significantly inhibit the growth of microorganisms, and reduce the contents of TVB-N and TBA. It was proved that the complex biological preservatives could effectively delay the deterioration of T. rubripes, prolonging the shelf life of 15 d to 30 d.