The invention discloses a gastric mucosa epithelial cell ATP4B promoter-driven simian virus 40 Tag (SV40T)-green fluorescent protein (GFP) eukaryotic expression vector. The gastric mucosa epithelial cell ATP4B promoter-driven SV40T-GFP eukaryotic expression vector comprises an ATP4B promoter sequence, a SV40T gene sequence, and a gene fusion recombinant plasmid of a vector plasmid DNA sequence with a GFP gene sequence. A construction method of the gastric mucosa epithelial cell ATP4B promoter-driven SV40T-GFP eukaryotic expression vector comprises the following steps of 1, cloning the ATP4B promoter sequence, 2, inserting the ATP4B promoter sequence cloned in the step 1 into the vector plasmid with the GFP gene sequence and carrying out verification, and 3, inserting the SV40T gene sequence into the vector plasmid with the ATP4B promoter sequence and the GFP gene sequence and carrying out digestion identification and sequencing. The gastric mucosa epithelial cell ATP4B promoter-driven SV40T-GFP eukaryotic expression vector does not damage cells, has a low cost, is convenient for use, provides a study object and lays an experimental base for further studying a stomach cancer occurrence mechanism and developing stomach cancer diagnosis and gene therapy.