Abstract: By hot water extraction method, we extracted the protein hydrolysate of tilapia wastes, which is used as a nitrogen source for L-lactic acid fermentation. Compared with yeast extract, we studied the growth of Lactobacillus bulgaricus, Streptococcus thermophilus and L.rhamnosus in fermentation medium of hydrolysate of tilapia wastes,and abservedtheyield of L-lactic acid. Results show that the 3 lactic acid bacteria grow better in 2 fermentation mediums and pH decreases obviously. The production of L-lactic acid varies with different lactic acid bacteria. The net yield of L-lactic acid is 3.38 gL-1 after 10 h fermentation with L.rhamnosus in the fermentation medium of hydrolysate of tilapia wastes. Theyield is considerable to that of the yeast extract. Therefore, the hydrolysate of tilapia wastes can be a substitute for yeast extract and is a low-cost nitrogen source for theyield of L-lactic acid.