Abstract: This study established a high-efficiency fermentation process for L-homoserine production using the threonine auxotrophic Escherichia coli strain HS27. The effects of key components in the fermentation medium on product synthesis were systematically investigated. It was demonstrated that the mass ratio of yeast extract powder to corn steep liquor powder, and the mass concentrations of beet molasses and ammonium sulfate significantly impacted L-homoserine production. Further optimization via orthogonal experimental design determined the optimal medium composition as follows: glucose 15.0 g/L, MgSO₄·7H₂O 1.5 g/L, yeast extract powder 0.6 g/L, corn steep liquor powder 2.4 g/L, beet molasses 10.0 g/L, L-threonine 0.4 g/L, (NH₄)₂SO₄ 3.0 g/L, KH₂PO₄ 1.5 g/L. Under these conditions, the highest shake-flask fermentation titer reached 7.46 g/L, representing a 19% increase compared to the initial conditions. Subsequently, a fed-batch process incorporating a two-stage threonine feeding strategy and betaine feeding was established in a 5 L multi-parameter fermentation bioreactor. By maintaining the oxygen uptake rate (OUR) at 150±5 mmol/L/h, the L-homoserine titer and synthesis rate were effectively enhanced. Ultimately, an L-homoserine titer of 134.1 g/L and an average synthesis rate of 4.8 g/L/h were achieved. This work lays an industrial foundation for the high-efficiency fermentation production of L-homoserine using the engineered E. coli strain HS27.