Objective A human hepatocellular carcinoma cell line (HCCLM3-Luc) stably expressing a luciferasereporter gene was used to establish an orthotopic mouse model. Methods An imaging system for living animals was used toquantitatively analyze the bioluminescence intensity of HCCLM3-Luc cells, which reflects the luciferase activity in cells.Then, the linear correlation between the photon quantity generated by HCCLM3-Luc luciferase and the number of HCCLM3-Luc cells was explored. The nude mouse model of orthotopic human hepatocellular carcinoma was constructed by in situinoculation of HCCLM3-Luc tumor tissue in the hepatic lobe or injection with HCCLM3-Luc cell suspension through the tailvein. The two methods were evaluated and compared based on the tumor formation rate, the time of formation and thechange of tumor shape. Results The bioluminescence intensity of the cells was positively correlated with the number ofcells (correlation coefficient R² = 0. 9989, linear equation: Y = 1155. 8X + 1× 10⁶ ), and the average ROI value wasapproximately 140 photons/ s per cell. Conclusions In this study, an orthotopic hepatocellular carcinoma-bearing mousemodel using a bioluminescent human hepatocellular carcinoma cell line was established. The tumor growth was assessed by measuring the bioluminescence in real time using the IVIS Lumina XR Imaging System.