Abstract: Objective To prepare monoclonal antibodies (mAbs) against multiple antigens by single cell fusion. Methods BALB/c mice were immunized with the multiple antigens, namely alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), HBsAg,HBcAg and HBeAg, and hybridomas were employed using PEG as the fusing agent. The hybridoma cells were respectively screened with AFP, CEA, HBsAg, HBcAg and HBeAg by enzyme-linked immunosorbent assay and limited dilution. The mAbs were purified by protein G affinity chromatography, its subtype was identified, the affinity constants (Ka) were determined and the specificity was analyzed by Western blotting. Results Twenty hybridoma cell lines were obtained by single cell fusion, including 5 cell lines against AFP, 6 against CEA, 3 against HBsAg, 4 against HBcAg, and 2 against HBeAg. The subtypes of some hybridoma cell lines positive for the mAbs were identified as the immunoglobulin G1 (IgG1), with Ka ranging from 1×10⁹ M^-1 to ×10¹¹ M^-1. Western blot analysis showed that all the mAbs strongly and specifically bound to their respective antigens. Conclusion The mAbs against multiple antigens have been obtained by single cell-fusion, which increases the production of mAbs and reduces the time of preparation.