The invention provides a separation and purification process of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside (short for stilbene glucoside). The separation and purification process comprises the steps of weighting a stilbene glucoside extractive, stirring a sample by using silica gel until the sample is loosened, eluting the loosened sample on a glass column by using an eluent, tracking and checking through a thin layer chromatography (TLC), merging a stilbene glucoside contained component, recovering a solvent, and drying to obtain the stilbene glucoside extractive; adding pure water, heating to dissolve the stilbene glucoside extractive, then, freezing and crystallizing the solution, carrying out suction filtration after the frozen and crystallized solution is molten, and washing the solution with the pure water to obtain a white crystal; transferring filter residues to a vacuum drying oven for drying to obtain a pure product of stilbene glucoside. The process is simple and feasible in operation, capable of improving the product purity, and capable of quickly separating and purifying a large amount of stilbene glucoside. The process is simple and convenient to operate, low in cost, stable in yield, capable of obtaining high-purity stilbene glucoside after re-crystallization treatment while keeping low cost and high yield, and suitable for meeting the requirements for large-scale industrial production.