The invention provides a phenotype maintaining and in-vitro high-activity three-dimensional amplification method of hepatic stem cells. The method comprises the following steps: 1, preparing a medium, wherein the medium comprises 1L of DMEM / F12, 15-20 mg / L of a fibroblast growth factor, 10-15 mg / L of insulin, 0.1-0.2 mg / L of glutamic acid, 0.5-0.8 mg / L of nicotinamide, 0.1-0.2 mg / L of seleninic acid, 0.05-0.1 mg / L of beta-mercaptoethanol, 0.1-0.6 mg / L of alpha-actin, 0.2-0.3 mg / L of sodium acetate, 0.001-0.003 mg / L of sodium pyruvate and 2-3 wt% of fetal calf serum; and 2, immersing sterilized porous gel in the medium, inoculating the porous gel with hepatic stem cells, and culturing the hepatic stem cells. Stem cells with stable phenotype and high activity are obtained in the invention, and the good characteristics of the stem cells are kept.