Objective To explore the construction of dipeptidase 1（DPEP1）gene expression vector，and to investigate the overexpression of DPEP1 protein in tumor cells and its impact. Methods Polymerase chain reaction was used to amplify the DPEP1 full-length gene，and EcoR V and Nhe I were used to perform the double-enzyme digestion. Then the digested products were cloned into the same double enzyme-digested pcDNA5/FRT/TO expres⁃sion vector to obtain pcDNA5/FRT/T0/DPEP1. The positive clones were screened out and verified using Sanger se⁃quencing. After that，the pcDNA5/FRT/T0/DPEP1 plasmid was amplified and transfected into cell line BT549 to ob⁃tain DPEP1 overexpression. Western blot，Cell Counting Kit-8，and other experimental techniques were used to an⁃alyze its expression and influences. Results The overexpression vector for DPEP1 was successfully constructed and transfected into the tumor cell line，and the DPEP1 overexpressed protein was obtained. The target protein had sig⁃nificantly up-regulated expression，and the overexpressed band had an intensity about 20 times that in the negative control. Conclusion The successful implementation of this study lays an experimental foundation for understanding the role and mechanism of action of DPEP1 gene in tumors in the future.