Nutrients such as L-threonine need to be added during the fermentation of L-homoserine by auxotrophic strains. In this study, a non-auxotrophic Escherichia coli chassis for the high-yield production of L-homoserine was constructed. The expression of thrB and thrC were down-regulated to attenuate the degradation of L-homoserine; then the overexpression of thrA, ppc and pntAB was performed to reinforce the synthetic flux and improve precursor supply and NADPH generation; finally, rhtA was overexpressed to elevate L-homoserine efflux. After 48 h fermentation in a 5 L fermenter, the constructed strain H-10 produced 52.1 g/L of L-homoserine with a percentage transformation of glucose of 43%, which is higher than the values reported in the literature.