In this work we have researched the effection of the substrates glucose, lactic acid, the mixture of glucose and lactic acid that were used by the immobilized propionibacteria on the fermentation of immobilized propionibacteria. The experiment results showed that when glucose was the substrate the optimum concentration was 40g/L and the production of propionic acid was 0.32g/L; the optimum concentration of lactic acid as the substrate was 40g/L and the production of propionic acid was 0.22g/L; the optimum concentration of mixture of glucose and lactic acid was 10g/Lglucose +40g/L and the production of propionic acid was 0.48g/L.The substrates that mixed by glucose and lactic acid were preferable to the single substrate on the production of propionic acid .