Abstract: Using uniform design, the optimal control model had been established for the fermentation of L-Arginine-¹⁵N₄ in 5 L fermentation tank. L-arginine was produced when the cell grew about 24 h. The theoretical yield can up to 22.49 g/L when production condition controlled in 820 r/min, pH 7.8, temperature in 30 ℃, the ventilation 2.5 L/min and fed-batch 60% glucose. While the actual yield was 88% of theoretical yield and 19.69 g/L of L-Arginine, which was verified. L-Arginine-¹⁵N₄ was produced by the abundance 99.68% of urea and 99.43% of ammonium sulphate, which ¹⁵N isotopic abundance was up to 98.7%, the purity was 98.5%. The ¹⁵N isotope abundance decreased less than 1%. The process was satisfied the request of batch production.