The broadly neutralizing antibody (bNAb) of human immunodeficiency virus (HIV) monotherapy eventually results in viral escape mutations in human. Bi- or multi-specific antibodies developed based on bNAbs show better neutralization potency and breadth. According to the published scFv gene antibody sequence, a bispecific antibody iMab-PGT151 encoded by a single gene was synthesized after codon optimization, and the recombinant plasmid was verified by double digestion and sequencing. Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding specificity of the bispecific antibody and the luciferase activity in the lysate of U87 cells to quantitatively analyze the neutralizing effect of the bispecific antibody on the HIV-1 pseudovirus，the indirect immunofluorescence test was used to detect the reactivity of the bispecific antibody iMab-PGT151 to human laryngeal carcinoma epithelial cells and the ELISA was used to detect the antibody's ability to bind cardiolipin and verify its autoreactivity. The results showed that the constructed bispecific antibody iMab-PGT151 could be successfully expressed; it could bind each ligand of the parent antibody separately and had bispecificity; iMab-PGT151 could neutralize 100% of 20 pseudoviruses with IC50 value of 0.084 μg/mL compared with the parent antibody; the antibody had stronger neutralization potency and breath without autoreactivity, and had clinical applicability. Bispecific antibody iMab-PGT151 may become one of the promising candidates for the prevention and treatment of HIV-1 infection in the future.