The invention discloses an isoelectric focusing electrophoretic method of hemopoietin. A BIO-RAD 111 type electrophoresis apparatus is used to carry out isoelectric focusing electrophoresis. Each 5 mLof glue comprises 1 mL of acrylamide monomer solution (25 wt%), 3.33 mL of urea (10.5 M), 0.412 mL of cane sugar (60wt%), 0.183 mL of amphoteric electrolyte solution (40 wt%, pH 2-4), 0.075 mL of amphoteric electrolyte solution (40 wt%, pH 3-10), and a catalyst solution, wherein the catalyst solution is mixed in advance and comprises 25 [mu]L of FMN-Na (0.1 wt%), 7.5 [mu]L of AP (10 wt%), and 1.5[mL] of TEMED. Before sample injection, pre-electrophoresis is performed for 10 minutes under a voltage of 200 V. Electrophoresis is performed through following phases: first phase, 50 V, 30 minutes;second phase, 100 V, 60 minutes; third phase, 200 V, 30 minutes; fourth phase, 450 V, 60 minutes; and fifth phase, 590 V, until the current is zero. The method can separate the hemopoietin charge isomers with different isoelectric points, and has the advantages that the requirement on the upper limit of voltage is not high and no cooling apparatus is needed.