Six levels of crude protein (0.21 to 3.36 g/L) were investigated as the nitrogen source for production of L-lactic acid by Rhizopus oryzae with respect to the influence on L-lactic acid yield, biomass and mycelia morphology. Increases in crude protein from 0.21 to 1.68 g/L led to an increase in L-lactic acid concentration from 6.48 to 57.7 g/L. However, further increases beyond 1.68 g/L did not present continuing increases in L-lactic acid yields. Fresh dairy manure was employed as a base substrate to produce L-lactic acid. The parameters, including glucose, dairy manure, spore culture time and etc, were first examined using Plackett-Burman design to select the most important ones. In the optimization stage, a factorial design (24) was carried out for the most important components. The main effects were summarized by a polynomial regression equation to describe the L-lactic acid production. The optimum medium composition was 3 g L-1 nitrogen, 120 g L-1 glucose, 1.3 g L-1 KH2PO4, and 0.5 g L-1 MgSO4, and the L-lactic acid yield was 29.1% (measured) and 30.76% (predicted), based on the experimental conditions used in this study.