A human hepatoma cell line, 2.2.15 cell, transfected with cloned HBV DNA, was used as a model to evaluate the inhibitory effects on HBV of the fermentation broth of Ganoderma lucidum with Radix Sophorae Flavescentis, a traditional Chinese medicine, as a medium component. At the same time, a mixture of the aqueous extract of Radix Sophorae Flavescentis and the fermentation broth of Ganoderma lucidum cultured in the basic medium without addition of Radix Sophorae Flavescentis, was also prepared and its inhibitory effects on HBV were compared with the model. The contents of hepatitis B surface antigen (HBsAg) and~( )core-related antigen (HBeAg) in the cell culture medium were measured with radioimmunoassay. The cytotoxinic effects on 2.2.15 cell were evaluated with MTT assay. The results showed that both methods reduced the contents of HbsAg and HBeAg in the cell culture medium, when adding dosage of the fermentation broth or the mixture ranging from 12.5 μg/mL to 200 μg/mL. The results also showed that the fermentation broth only had slight cytotoxinic effects on the cell, while the mixture had stronger cytotoxinic effects on the cell. The results indicated that the fermentation broth of Ganoderma lucidum with Radix Sophorae Flavescentis addition had an inhibitory effect on HBV in vitro.