Ribavirin and lamivudine are representatives of antiviral drugs that are widely used to treat viral infections, especially chronic liver disease. To compare binding mechanism and behavior of antiviral drugs with human serum albumin (HSA), we performed fluorescence spectroscopy and X-ray crystallography to investigate the interactions of ribavirin and lamivudine with HSA. Fluorescence spectroscopy showed ribavirin and lamivudine inhibit binding affinity each other. Our results further demonstrated that ribavirin and lamivdudine interaction with HSA could be affected by the presence of other compounds, including the non-steroidal anti-inflammatory drugs, indometacin. X-ray structures revealed that ribavirin and lamivudine bind in IIA subdomain of HSA mainly by forming hydrogen bond and hydrophobic interactions forces. The carboxamido of ribavirin forms hydrogen bonds with Arg222; Hydroxyl group (6) of ribavirin forms hydrogen bond with Arg257. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222; amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257. Our results reveal the key biochemical and structural characteristics of the HSA interaction with ribavirin and lamivudine, providing guidance for future development of ribavirin- and lamivudine-based compounds and a drug-HSA delivery system.