The invention relates to a preparation method of hydroxyl-terminated hyperbranched polyurethane. The operation steps include that 1 hydroxyl-terminated micromolecule polyhydric alcohol is dissolved in a non-proton organic solvent to obtain a hydroxyl-terminated micromolecule polyhydric alcohol solution; 2 the temperature of diisocyanate is raised to 70-90 DEG C, the hydroxyl-terminated micromolecule polyhydric alcohol solution is added, catalyst is added, and a reaction is conducted to obtain first generation hyperbranched prepolymer a1; and 3 the temperature of the first generation hyperbranched prepolymer a1 and di-alkanolamine is reduced to 0-15 DEG C, the temperature is kept, a reaction is conducted after blending, and products are separated and purified through precipitator and vacuum-dried to obtain first generation hydroxyl-terminated hyperbranched polyurethane A1. A synthesizing process is good in controllability, and the synthesized hyperbranched polyurethane is high in purity and neat in structure, contains large amount of terminated hydroxyl and is suitable for being used as a biological medicine carrier, a cross-linking agent, a viscosity modifier, a thermosetting material curing agent, a phase solubilizer, a rheological agent, thixotrope, a nucleating agent and the like.