Abstract: In order to avoid spoilage caused by microbial contamination during cold beef storage,and prolong the shelf life of cold beef,cold fresh beef was used as the research object. The minimum inhibitory concentration of preservative and the synergistic effect between the two natural preservatives were measured by double dilution method and checkerboard dilution method. Four natural preservatives were screened out. The TVB-N value was used as the detection index,and the cold beef treated with sterile distilled water was used as the control group. The four-factor and three-level response surface experiments were used to optimize the four compound natural preservatives and treated with no preservative. The cold beef was used as a control,and the best compound natural preservatives to the preservation effect of chilled beef was studied. The results showed that the optimal formula was polylysine 109.5 mg/kg,nisin 258.0 mg/kg,tea polyphenol 152.0 mg/kg,chitosan 130.0 mg/kg. It estended the cold beef’s shelf life from 6 to 18 d. This experiment screened and optimized the compound preservative to provide a technical reference for the rational use of cold beef preservative.