Fibronectin (FN) is an important molecule that participates in hepatitis B infection and fibrosis. Lumbrokinase (LK) is synthesized by earthworms containing a group of proteolytic isozymes, which were used to hydrolyze fibrin in the treatment of clotting disease. One of the isozymes purified from the LK compound was verified as a hydrolase to degrade FN in vitro and was called earthworm fibronectin hydrolase (EFNase). However, whether LK functions to prevent the deterioration of hepatitis B virus (HBV) infection has not been clarified. In this study, we used HepG2.2.15 cells to investigate the effect of LK on the level of hepatitis B surface antigen (HBsAg) or FN, used C57BL/6J-HBV transgenic mice to explore the effect on the level of HBsAg, FN, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, the pathological changes in livers. The results indicated that LK could inhibit HBsAg production in vitro and in vivo; decreased FN in serum and liver. Compared with physiological saline, LK improved the state of the livers. Those data provided valuable information to understand the therapeutic role of LK as a potentially effective medicine to treat chronic hepatitis B (CHB).