Abstract: Objective To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 (hTFF2) fusion gene to prepare for genetic modification of Lactococcus lactis that can secrete bioactive c-myc-hTFF2 protein. Methods Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5’ ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk (+), the SalI and BamHI sites were arranged at the 5’ and 3’ ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript II sk (+) to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with BamHI/SalI and of pNBC1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion. Results and Conclusion The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium of Lactococcus lactis.