The invention provides a low-cost establishment method of a virus in-vitro three-dimensional culture model. The establishment method comprises the following operating steps: (1) preparing a micro-carrier suspension; (2) preparing a cell adherent retardant; and (3) preparing a six-pore plate; uniformly paving the prepared cell adherent retardant in the six-pore plate; keeping and air-drying the six-pore plate; then, inoculating the six-pore plate with prepared cells by a concentration of 10<5>/ml, and adding the prepared micro-carrier suspension, wherein the ratio of cell liquid to the micro-carrier suspension is at 4: 1; uniformly mixing the cell liquid and the micro-carrier suspension; keeping an obtained mixture in a cell incubator; and implementing a virus inoculation experiment as a cell three-dimensional structure is formed after 3-4 days. According to the method, high cost caused by an animal in-vivo experiment can be avoided, and meanwhile, an animal in-vivo growth model can be simulated to the greatest extent, so that the cultivated cells follow a three-dimensional growth mode, and a similar in-vivo tissue structure is formed; therefore, the establishment method is beneficial for simulating a virus in-vivo pathogenesis process; and the model establishment method is simple, low in cost and convenient for popularization.