Objective Lumbrokinase isozymes (LKIs), which were isolated from earthworm called Dilong in traditional Chinese medicine (TCM), have been used as the active ingredient of the enteric-coated capsule to treat clotting disease approximately 30 years. Recently, the study of LKIs on other critical diseases received much attention. Methods To demonstrate the efficacy of LKIs on hepatitis B proteins, we incubated surface antigen (HBsAg), core antigen (HBcAg) and e antigen (HBeAg) with LKIs at different concentrations for different time intervals, and then estimated their cleavage sites. HepG2.2.15 cells were incubated with LKIs and their HBsAg, HbeAg were determined by ELISA and Western blotting. HBV-transgenic mice (Balb/c) were gavaged with LKIs for 30 days. HBsAg and HBeAg in serum were detected by ELISA and Western blotting, and HBcAg in hepatic tissues were immunohistochemically stained. Hematoxylin-eosin (HE) staining was used to exhibit liver endolysis of LKI-treated HBV-transgenic mice. Serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were semi-quantitatively detected with ELISA. After intraperitoneal injection of LKIs into Sprague Dawley rats, LKIs in serum and liver tissue were assayed. Longyan sheldrakes (LYS) were gavaged with LKIs for 30 days, and their serum HBV DNA were assayed by PCR. Results We observed that the capsule ingredient is a compound drug containing 6 LKIs. By incubating with the HBV proteins, LKIs were probably estimated to degrade HBsAg at K141/P142 and R160/F161, HBcAg at R142/E143, and HBeAg at R122/E123. LKIs significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells. Levels of HBsAg and HBeAg in serum and those of HBcAg in hepatic tissues decreased in HBV-transgenic mice gavaged with LKIs, suggesting a suppression of viral assembly. Levels of GOT and GPT and the number of endolysis in liver exhibited by HE staining were decreased in the LKI-treated HBV-transgenic mice, demonstrating LKIs’ protecting mice hepatic cells. The activity of LKIs could be detected in serum and hepatic tissues of Sprague Dawley rats after being intraperitoneally injected with LKIs. After gavaged with LKIs, the ducks showed a decrease in their serum HBV DNA levels. Conclusion The current work indicates that LKIs degrade HBs, HBc and HBe proteins and may interfere with the virion assembly and release, leading to decrease in the virus transmission between hepatocytes, and to hepatic protection.