The invention discloses a method for separating and purifying protein by combining reverse micelle extraction with a pulsed electric field. The method comprises the following steps: (1) adding a target protein raw material into a reverse micelle solution, evenly mixing and then transferring to an extraction cavity of the pulsed electric field, wherein the extraction cavity of the pulsed electric field is arranged in a cooling tank, and implementing pulsed electric field combined with reverse micelle extraction; (2) carrying out solid-liquid separation on the solution, treated in the step (1),in the extraction cavity of the pulsed electric field, taking supernatant to obtain a pre-extracted solution of the target protein; (3) carrying out back extraction on the obtained pre-extracted solution to obtain post-extraction liquid containing the target protein; (4) concentrating and drying the post-extraction liquid to obtain the target protein. The method can increase the extraction yield of the protein and shorten the extraction time; furthermore, the quality of the product is also obviously superior to that of products obtained by using the traditional extraction method.