Abstract: Objective: In this study, Corynebacterium glutamicum ATCC 13032 was used as the chassis cell for synthesizing L-homoserine and analyzing the effect of dissolved oxygen on product synthesis. Methods: First, the product tolerance of C. glutamicum was analyzed by exogenously adding 0~40 g/L L-homoserine. Second, the degradation pathway of L-homoserine was blocked by gene thrB knockout, namely C. glutamicum recombinant strain H1. On this basis, the shake flask with baffles was used for cell culture to enhance oxygen supply capacity in the fermentation process. Results: Compared with Escherichia coli, C. glutamicum had a stronger tolerance to L-homoserine. In the study, C. glutamicum recombinant strain H1 was constructed by deleting the gene thrB. It was found that the growth of recombinant strain H1 returned to normal after adding 0.5 g/L L-threonine in the basal medium. The L-homoserine production of recombinant strain H1 increased to 836.7 mg/L using shake flask with baffles, which was 17.76 times higher than that using ordinary shake flask, which was 44.6 mg/L. Conclusion: C. glutamicum recombinant strain H1 was successfully constructed for producing L-homoserine via blocking the synthesis of L-threonine. It was found that the using of shake flask with baffles to enhance the oxygen supply capacity during fermentation was an effective means to promote the production of L-homoserine by C. glutamicum. This study provides a reference for improving L-homoserine production subsequently.