Abstract: Objective To construct the pEGFP-N1-ALDH1A1 eukaryotic expression vector and evaluate its biological function in human gastric cancer cell line MKN-28. Methods A cDNA encoding ALDH1A1 was cloned by gene cloning techniques； The pEGFP-N1-ALDH1A1 was constructed by use of recombinant DNA technique and was demonstrated by restriction endonuclease mapping and sequencing.The pEGFP-N1-ALDH1A1 was transfected into human gastric cell line MKN-28 by using lipofectamine 2000.The protein expression of ALDH1A1 was detected by the Western blotting assay. The activity of ALDH was detected with ultraviolet-visible light detector. Results A cDNA encoding ALDH1A1 was successfully cloned；Western blotting showed the level of protein expression of ALDH1A1 in gastric cancer cell line MKN-28 transfected with pEGFP-N1-ALDH1A1 were significantly increased，and the ALDH1A1 activity in MKN-28 transfected with pEGFP-N1-ALDH1A1 were significantly increased. Conclusion The recombinant plasmid of pEGFP-N1-ALDH1A1 was constructed corrected and the protein of ALDH1A1 overexpressed in human gastric cancer cell line MKN-28. It provided solid experiment foundation for the further studies on the function of ALDH1A1 in gastric cancer.