Objective To observe the effect of artificially designed and chemically synthesized polypeptides on HBV-DNA replication and expression of viral markers, as well as their cytotoxicity, screen polypeptide of high inhibition and low cytotoxicity HBV, and explore the potential of polypeptides as novel antiviral molecules for HBV. Methods Seven polypeptides (KBDT-1, 2, 3...7 based on hydrophobicity and cationic electroaffinity) were designed and synthesized using traditional methacrylic acid polymer platform, seven chemically synthesized polypeptides (10 mg/mL), lamivudine (1 mg/mL, positive control) and blank solvent (negative control) were applied to HepG 2.2.15 cell line, the inhibition effect of chemically synthesized polypeptides on HBV was detected, cell viabi-lity rate was detected by crystal violet staining, cytotoxicity of each group was compared. Polypeptide with the strongest inhibition effect on HBV was selected, concentration gradients of 10, 1 and 0.1 mg/mL were set, after 3, 6 and 9 days of treatment on HepG 2.2.15 cells, the supernatant of cells was collected, copies of viral DNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR), changes in HBsAg and HBeAg were detected by chemiluminescent microparticle immunoassay. Results Peptide KBDT-2 was screened from seven chemically synthesized polypeptides, RT-PCR result showed that KBDT-2 had anti-HBV replication effect in vitro, and the higher the concentration of KBDT-2, the better the inhibition effect; chemiluminescence microparticle immunoassay showed that KBDT-2 could inhibit HBsAg and HBeAg, the biological markers of hepatitis B virus; crystal violet staining result showed that KBDT-2 had no obvious toxicity to HepG 2.2.15. Conclusion KBDT-2 can inhibit the replication of HBV without obvious cytotoxicity, and can effectively reduce the expression of HBsAg and HBeAg, which provides experimental data support for exploring new anti-HBV drugs.