In this research, the fermentative production of D-lactic acid from glucose by engineered Escherichia coli was examined. Herein, the D-lactic acid production by Escherichia coli expressing recombinant α-amylase and β-glucosidase was reported, using starch and cellobiose, respectively, as its sole carbon sources. By performing cell surface display technique, pGV3-SBA and pGV3-bglC plasmids containing amyA and bglC genes from Streptococcus bovis NRIC 1535 and Thermobifida fusca YX, were respectively transferred into Escherichia coli JM109 to test the ability to produce α-amylase and β-glucosidase for starch and cellobiose degradation. After extraction of the plasmids containing target genes from the E. coli transformants, the DNA electrophoresis gel showed bands at 9 kb and 8 kb corresponding to pGV3-SBA and pGV3-bglC plasmids, respectively. The transformation of E. coli BW25113 inactivating pflA gene with the respective plasmids resulted in the ability of the transformants to survive in LB medium supplemented with spectinomycin and gave bands on SDS-PAGE gel at 85.3 kDa and 53.4 kDa proteins corresponding to recombinant α-amylase and β-glucosidase, respectively. By cultivation of the recombinants in LB medium and induction of gene expression by addition of 1 mM IPTG, specific enzymatic activities of α-amylase and β-glucosidase were obtained as 23.13 U/OD₆₀₀ and 18.90 U/OD₆₀₀, respectively. In M9 medium supplemented with starch and cellobiose as the sole carbon sources, E. coli­ BW25113 (∆pflA, pGV3-SBA) and (∆pflA, pGV-bglC) could produce D-lactic acid at 1.67 g/L and at 2.59 g/L, respectively, with 100% purity in 48 h under static culture condition. These results supported for the idea that starch and cellobiose can be used as cheap carbon sources for D-lactic acid production.