In this study, dairy manure as a nitrogen source was evaluated for L-lactic acid fermentation by Rhizopus oryzae. (NH₄)₂SO₄ in culture media was replaced with the purified crude protein from dairy manure (appropriate nitrogen concentration was 0.42 g/L for Rhizopus oryzae based on (NH₄)₂SO₄). Six levels of nitrogen were used, 0.21 g/L, 0.42 g/L, 0.84 g/L, 1.68 g/L, 2.52 g/L, and 3.36 g/L and the results showed that the corresponding L-lactic acid yields were 6.48 g/L, 14.73 g/L, 38.33 g/L, 55.7 g/L, 54.9 g/L, and 54.1 g/L, while it was 57g/L for control. To improve the utilization rate, crude protein hydrolysis was also studied. Results showed that the greatest hydrolysis degree (DH, 48.9%) was achieved at 0.06 g Alcalase/g of protein, pH 8.0, 53°C for 240 min. Six levels of diluted hydrolysates (same as above) were experimented and 55.9 g/L L-lactic acid was obtained using the hydrolyzed protein at 0.42 g/L and ≥ DH 33.8%. A uniform design (U₆ (6²×3)) was applied to optimize seed culture using three factors (nitrogen and spore concentration, treatment durations). It was observed that diameter of the seed pellet was 1.03 ± 0.12 mm after 20 hours incubation with optimal medium containing 1.68 g/L nitrogen and 1 ×10⁶ spore/mL. A second uniform design (U₈ (8⁵)) with five factors (glucose, nitrogen in dairy manure, ZnSO₄, KH₂PO₄ and MgSO₄) was also experimented to optimize flask culture, showing lactic acid yield was 60.5% by weight, 8% higher than the control.