In this study, we constructed a recombinant Escherichia coli strain for the production of high-purity L-lactic acid, using a homoethanol fermenting mutant E. coli SZ470 (deltafrdBC deltaldhA deltaackA deltafocA-pflB deltapdhR: :pflBp6-pflBrbs-aceEF-lpd) as the starting strain. By using homologous recombination, we deleted the adhE gene from SZ470 to obtain a mutant Escherichia coli JH01, which could not grow under anaerobic conditions. Then we cloned the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici and inserted it into the chromosome of JH01 via electroporation to obtain a recombinant strain Escherichia coli JH12. We evaluated the L-lactic acid production of the recombinant strain in a 15 L fermenter. In 10 L LB medium supplemented with 6% glucose, JH12 maintained maximal cell growth and an efficient L-lactic acid production rate for 36 h. Glucose consumption rate achieved was 1.46 g/(L x h) and L-lactic acid production rate was 1.14 g/(L x h). The results also show that 41.13 g/L lactic acid was produced, achieving a purity of 95.69% (based on total fermentation products). Xylose consumption rate was 0.88 g/(L x h) and L-lactic acid production rate was 0.60 g/(L x h). The production of lactic acid was 34.73 g/L, achieving a purity of 98%. There were no succinic acid and formic acid detected and only little amount of acetic acid generated during the fermentation. We constructed a homolactic acid fermentation strain E. coli JH12, which could efficiently convert glucose and xylose into high-purity L-lactic acid. JH12 could have great potential in industrial fermentation for L-lactic acid production.