The bacteriostasis effect of biological preservative such as lysozyme, nisin, chitosan, tea polyphenols and cinnamon essential oil on chilled beef was studied. The experimental results showed that chilled beef treated with no preservatives at room temperature deteriorated on ninth day, but the chilled beef treated with preservative treatment deteriorated on fifteenth day. The regression equation was established based on response surface methodology to analyze the total colony counts of chilled beef on the eighteenth day after mixed treatment with 3 optimal preservatives obtained by single factor experiment. The antistaling agent had the optimal preservation effect with the mass fraction of 0.75% chitosan, 1.29% tea polyphenols and 2.91% lysozyme, while the maximum number of lg（N/（CFU·g-1））was 5.61, which was lower than the 6.00 prescribed by the state.