The invention discloses a method for performing high performance size exclusion chromatography (HPSEC) and purifying recombinant human proinsulin renaturation. The method comprises the following steps: (1) performing ultrasonic cell disruption on the proinsulin-containing thallus cell, washing and dissolving the buffer solution in strong denaturant (such as 8mol/L or 6-7 mol/L guanidine hydrochloride) thereby obtaining the recombinant human proinsulin subjected to crude separation; (2) directly feeding the proinsulin-containing denaturated leaching liquor under the optimized chromatographic condition, wherein the purity of target fraction rhPI obtained through one-step chromatography by employing urea concentration linear gradient elute can be over 96 percent, and the mass recovery rate is 56.8 percent; and (3) further desalting the proinsulin-containing original fraction, thereby obtaining the renaturated and purified high-purity and activity fraction. The recombinant human proinsulin obtained by the method can be converted into insulin through digestion on a chromatographic column or digestion in a solution, and the chromatographic process is easy to operate, high in repeatability and easy for large-scale production.