L-cysteine is a ubiquitous and unique sulfur-containing amino acid with important physiological functions. The efficient L-cysteine production via microbial fermentation is interesting and has been paid great attention. In this study, different Escherichia coli K-12 strains (JM109, BW25113, MG1655, W3110) were investigated on their suitability to cysteine-producing plasmid pLH03. The enhancement of precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of BW25113. The expressions of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on L-cysteine production. Main degradation pathway genes were also deleted coordinately for more efficient production of cysteine. The L-cysteine production was further increased through the manipulation of sulfur transcription regulator cysB and sulfur supplement. After the process optimization in a 1.5 L bioreactor, the final engineered strain LH2A1M0B△YTS-pLH03 [BW25113Ptrc2-serA-Ptrc1-cysM- Ptrc-cysB△yhaM△tnaA△sdaA-(pLH03)] accumulated 8.34 g/L of cysteine, laying a certain foundation for cysteine fermentation industry.