Abstract: In this study,Lactobacillus plantarum ALAC- 3 and ALAC- 4 being of antifungal activity was chosen to produce Cheddar cheese combining with industrial fermentation agent.Single factor experiment and orthogonal experiments were used to traditional strain and optimize ratio of different lactobacillus strains with fungal inhibition,inoculation amount,culture temperature and fermentation time. The antifungal effect of cheese adding the Lactobacillus was studied.The results showed the optimum fermentation conditions for ALAC-3,were as follows:the mixing proportion of traditional strain and ALAC-3 was 4∶ 4∶ 2.5,inoculum size was 3%,fermenting at 35 ℃ for20 min.For ALAC-4 the result was as follows: the mixing proportion of traditional strain and ALAC-4 was 4 ∶ 4 ∶ 0.5,inoculum size was 3%,fermenting at 37 ℃ for 25 min. Comparing with the traditional cheese,the special cheese products adding ALAC-3( ALAC- 4) had significantly antifungal effect when were stored at 25 ℃. Therefore,the Lactobacillus of ALAC- 3 and ALAC- 4 could be used as biological preservative in the production of cheddar cheese.