Abstract: In order to optimize an effective combined preservative applied to the orange juice preservation, the authors studied the synergistic effect of biological and chemical combined preservative which contained Nisin, potassium sorbate, EDTA and sodium isoascorbate in preservation of orange juice compared to preservation effect of each aforementioned additive in individual use. Results show that the growth of microorganisms can not be inhibited completely using each aforementioned additive in single use. Results of orthogonal experiment show that the growth of microorganisms can be significantly inhibited when the optimal composition of biological and chemical combined preservative contains 0.05 g/kg of Nisin, 0.1 g/kg of potassium sorbate, 0.01 g/kg of sodium isoascorbate and 0.03 g/kg of EDTA. The inhibitory rates in the 14th, 17th and 21st day were determined as 99.9%, 99.9% and 99.6%, respectively, the inhibitory effect of which is significantly better than that of any aforementioned preservative in single use. Therefore, cooperative and synergistic action can be demonstrated when Nisin and other three materials are added into orange juice for combined use. This combined preservative has good preservation effect on orange juice so that it has a broad application prospect.