Objective To obtain the expression vector, which could be used for screening peptide drug against human immunodeficiency virus type 1 (HIV-1) integrase (IN) with bimolecular fluorescence complementation (BiFc). Methods Full-length IN sequence was amplified using high-fidelity PCR with the template pMDL vector, following with the insertion of target sequence into pBiFc-VN173 vector. Moreover, the recombinant vector pBiFc-VN173-IN was further confirmed by double enzyme digestion and sequencing. Compared with empty control, expression of IN from pBiFc-VN173-IN in HEK293T cells was validated by Western blotting and immunofluorescence assay (IFA). Results The pBiFc-VN173-IN vector, which could drive the ectopic expression of IN, was successfully obtained through high-fidelity PCR, vector construction and confirmation. In addition, Western blot analysis and IFA validated the ectopic expression of IN in HEK293T cells after transfection. Conclusion The pBiFc-VN173-IN vector has been successfully obtained, and it will be helpful for screening specific peptides against IN using BiFc.