Abstract: In this study, the three dimensional(3D)organoid culture system was established by liquid overlay method, and applied as an effective model to evaluate the hepatic injury of susceptible compounds in Polygonum multiflorum Thunb. Compared with the ordinary two dimensional(2D)culture of liver cells, the albumin expression of L02 cells and HepG2 cells were increased by 2.5 and 6.7 times in the 3D organoid culture system, respectively. After the cultivation of 21 days, urea generation levels of 3D culture were increased by 8.3 and 15.5 times. More importantly, HepG2 cells were more suitable to development of organoids than L02 cells. The gene expressions of phase I and II drug metabolism enzymes of HepG2 cells cultured as 3D organoids were significantly increased than that in 2D culture, such as the fold changes of CYP2C9 was up to 381.9, CYP3A4 to 87.0, CYP2D6 to 312.6. In addition, drug transporter relative genes were also up-regulated. The results demonstrated that the liver synthesis and metabolic function of the 3D model were better than that of the 2D cultured hepatocytes. The results of hepatotoxicity evaluation showed this developed model can be used to assess the hepatotoxicity of acetaminophen and other positive control drugs, which were considered with defined hepatotoxicity. On the 3D culture model, the IC₅₀ value of repeated drug dose administration was significantly lower than that of single dose administration. However, the IC₅₀ of 2,3,5,4'-tetrahydroxy-cis-stilbene-2-O-β-glucoside(cis-SG), which is the susceptible compound in Polygonum multiflorum Thunb., could not be detected in 2D cultured model. With the treatment of a single dose administration in organ 3D culture model, the IC₅₀ of cis-SG was 1.9 times than that of cyclosporine A, and the IC₅₀ of 2,3,5,4'-tetrahydroxy-trans-stilbene-2-O-β-glucoside(trans-SG)was 4.1 times than cis-SG. The hepatotoxicity results of cis-SG and trans-SG on the 3D cultures were similar to in vivo toxicity results obtained in previous work. On organ 3D culture model, the IC₅₀ of cis-SG with repeat of administration decreased compared with that with single dose administration, suggesting that long-term medication may increase the risk of liver injury. In summary, the 3D organoid culture system can be used for a long period to preserve the capacity of liver synthesis and metabolism. The organoids were a model suitable for evaluation of mechanism of the drugs with low toxicity.