<p>Hepatitis B virus (HBV) is a pathogen that often causes progressive liver disease, leading to acute, chronic hepatitis, cirrhosis, and ultimately the development of hepatocellular carcinoma. Although several antiviral drugs, including interferon alpha and nucleotide derivatives, have been approved for clinical treatment of HBV, key issues such as low to moderate efficacy, adverse side effects, and drug-resistant strains remain unresolved. In this study, based on the results of molecular docking experiments, twenty matrine-adenine hybrids were synthesized and their antiviral effect against HBV was evaluated. Biological evaluations revealed that these synthesized compounds showed significant anti-HBV activity. Of these, ten compounds significantly inhibited HBsAg secretion more than the positive control lamivudine (3TC). Remarkably, compounds <b>4c</b>, <b>4d</b>, and <b>4k</b> showed the most significant inhibitory activity against HBV DNA replication with IC₅₀ value of them were 12.51 ± 1.24 to 24.10 ± 0.56 μM, respectively. Which is stronger than that of substrate MMA (IC₅₀ = 52.18 ± 3.14 μM). Structure–activity relationship (SAR) data concluded that with the growth of carbon chains in straight chain aliphatic hydrocarbons significantly enhanced anti-HBV activity, whereas cyclic aliphatic hydrocarbons substitutions contributed to moderate activity. Overall, this study identifies matrine–adenine hybrids as promising active compounds against HBV in vitro, providing a basis for further evaluation in more physiologically relevant models.</p> Graphical abstract <p></p>