The invention discloses an extraction method of thiol protease. The extraction method comprises the following steps: (1) mixing tapioca starch magnetic microspheres with a solution containing thiol protease, and then performing oscillating adsorption on the mixed liquid; (2) performing magnetic separation on the reaction liquid obtained in the step (1), and performing eluting on the tapioca starch magnetic microspheres so as to obtain the thiol protease. The separation method disclosed by the invention is simple to operate, short in time, simple in equipment requirements, and high in equilibrium absorption capacity.