L-alanine is one of the smallest chiral compounds and has been widely used in food， medicine， and daily chemical fields. Microbial production of L-alanine has problems such as long fermentation period and low productivity. In this study， metabolic engineering strategies such as strengthen precursors supply， promoter engineering and transporter engineering was used to construct an Escherichia coli cell factory with high-titer production of L-alanine. The production process of L-alanine was optimized by biochemical engineering strategies to improve the production performance of the Escherichia coli cell factory with production of L-alanine. Results: Overexpression of gapA(Glyceraldehyde 3-phosphate dehydrogenase gene) enhanced precursor supply， increased the titer of L-alanine and the conversion of glucose by 5.1% and 15.6%， respectively. Through the optimization of gapA expression by promoter engineering， the titer of L-alanine and the conversion of glucose reached 18.3 g/L and 0.55 g/g， respectively. Overexpression of L-alanine transporter (AlaE) enhanced L-alanine transport， the titer of L-alanine reached 20.4 g/L. The composition of culture medium was optimized by biochemical engineering strategy， the best carbon source was glucose 40 g/L and the best nitrogen source was (NH4)2SO4 25 g/L. The optimum fermentation conditions were as follows: inoculum size: 15%， fermentation mode: 10 h conversion to anaerobic fermentation and feeding mode: variable speed feed. After 36 h fermentation in a 5 L bioreactor， the constructed strain E. coli W-135 produced 127.2 g/L of L-alanine， with a yield of 0.83 g/g glucose and a productivity of 3.53 g/L/h， which were 64.3%， 50.9% and 64.2% higher than those before optimization， respectively. In this study， a L-alanine high-titer strain with short fermentation period and simple fermentation process was constructed by using the strategy of system metabolic engineering and biochemical engineering， which provided a theoretical basis for the industrial production of L-alanine.