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 observed the yield 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 g · L^-1 after 10 h fermentation with L.rhamnosus in the fermentation medium of hydrolysate of tilapia wastes. The yield 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 the yield of L-lactic acid.