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AIM: To explore an ideal method for extracting protoporphyrin disodium (PPN) from unanticoagulated animal blood, and to study the inhibitory effects of PPN on HBV-DNA duplication and its cytotoxicity to 2.2.15 cell strain.METHODS: Protoporphyrin methyl ester and other intermediate products were prepared with protoheme separated from protein hydrolysates of coagulated animal blood, which were finally made into PPN and detected quantitatively with an ultraviolet fluorescent analyzer.Ten μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml and 160 μg/ml of PPN-aqueous solution were added into culture medium for 2.2.15 cells respectively. Eight days later, the drug concentration in supatant from the culture medium was detected when inhibition rate of HBeAg, cell Survival rate when inhibition rate of HBeAg was 50% (ID50), and when survival cells in experimental group were 50% of those in control group (CD50), and the therapeutic index (TI) was also detected. PPN with different concentration of 10 μg/ml,20 μg/ml, 40 μg/ml, 80 μg/ml and 160 μg/ml was respectively mixed and cultivated with HepG2 2.2.15 cell suspension,and then the inhibition of PPN against HBV-DNA was judged by PCR.RESULTS: The extract of henna crystal was identified to be PPN. When the concentrations of PPN were 160 μg/ml and 80 μg/ml, the inhibition rates of HBeAg were 89.8% and 82.4%, and the cell survival rates were 98.7% and 99.2%.CONCLUSION: It is suggested that PPN can be extracted from unanticoagulated animal blood. PPN can inhibit HBV-DNA expression and duplication ih vitro, and has no cytotoxicity to liver cells. Further study and application of PPN are warranted.