The highest-efficiency preservatives and best mixed preservatives were screened by Oxford cup test using single factor test design and response surface test design, and Bacillus licheniformis and Bacillus LAMI 002, which were separated from spoiled eggs were tested as inhibition objects. The results showed that ε-polylysine, lysozyme and nisin could effectively inhibit the growth of spoilage bacteria. LAMI 002, and had a strong inhibitory effect on the growth of Gram-positive bacteria. The optimal formulation of preservatives was determined by response surface methodology as follows: Lysozyme 0.185 2 g/kg, ε-polylysine 0.054 1 g/kg and nisin 0.066 3 g/kg. In the verification test, the mixed putrefaction bacteria were obviously controlled effectively, and the total number of colonies was reduced by 99.07%.