Objective To isolate and identify primary hepatocytes from hepatitis B virus (HBV) transgenic mice, and provide a useful model for the study of HBV in vitro. Methods HBV transgenic C57 BL/ 6 mice were anesthetized and primary mouse hepatocytes (PMHs) were isolated and purified via a two-step infusion method. First, the liver was flushed with a calcium-free infusion, then digested with calcium-containing type IV collagen buffer. Hepatocytes were purified by centrifugation at low-speed and low-temperature three times, and the purified PMHs were confirmed by glycogen staining. In addition, the viability of PMHs within 7 days and the apoptotic ratio on days 1 and 3 were detected. Furthermore, PMHs were used to detect the effects of liver injury induced by hydrogen peroxide, including intracellular reactive oxygen species, mitochondrial membrane potential, oxidative stress factor level detection, and lactate dehydrogenase ( LDH) leakage. Results (1) The PMHs isolated from HBV transgenic mice showed typical hepatocyte morphology and positive glycogen staining. (2) The viability of hepatocytes increased after being adhered to a plate, and peaked at days 5 to 6, and then decreased gradually. (3) There was no difference between the apoptosis ratio on day 1 and day 3, with no spontaneous death. (4) Hydrogen peroxide had serious effects, including reactive oxygen accumulation, a loss in mitochondrial membrane potential, upregulation of oxidative stress factors, and LDH leakage. Conclusions Taken together, these data indicate that the PMHs of HBV can be successfully isolated and applied to liver injury research.