Abstract: On the basis of single factor experiment,the aim was to study the influence of three kinds of compound biological preservative on effect of cold preservation of P. vannamei through the analysis of Box-Behnken central composite design and response surface methodology(RSM). Using the total number of bacteria of P. vannamei which had been cold stored for 6 days as evaluation index,and using Design-Expert software to analyze the quadratic regression model of compound biological preservatives and the total number of bacteria,and then optimal concentration combinations of three kinds of compound biological preservative were obtained.The results showed that the optimal concentration of the three kinds of compound preservative were Nisin0.04g/100 m L,ε-PL 0.48g/100 m L and TP 1.06g/100 m L. The total number of bacteria was 1.81 ×103CFU/g. The largest number was 1.98×103CFU/g in the three parallel verification of total bacteria tests,error was only 9.39%,which meant the model was appropriate and credible. It could be used to analyze and predict the total number of bacteria of the P. vannamei when it was cold stored. The compound biological preservative could be mutually complementary and synergistic antibacterial,which could extend the shelf life of P. vannamei effectively.