Objective To define the best dose of PMSG/hCG for superovulation and optimize the biological purification system for genetic-modified mice of C57BL/6J background, we injected different dose of PMSG/hCG to promote superovulation, and carried out in vitro fertilization and embryo transplantation. Methods 5-week old female C57BL/6J mice were injected with 4 different doses(5 IU,7.5 IU,10 IU and 15 IU, respectively)of PMSG/hCG. Then the mice were mated with wild type male mice and embryos were obtained by cesarean section. In another approach, oocytes of superovulated mice were fertilized in vitro and then transplanted into surrogate female mice. Results In the cesarean section group, the highest rate of pregnancy was 89.00±19.05% in the 5 IU injection group. But the difference compared with other groups was not statistically significant. In the embryo transplantation group, the average number of oocytes from 10 IU injection group was 30.33±0.89, and the average number of 2-cell embryo was 23.78±0.19, significantly higher than those in the other three groups. Conclusions Administration of 5 IU PMSG/hCG will improve mating efficiency in cesarean section-based purification of C57BL/6J mice. 10 IU PMSG/hCG is the best dose for superovulation of C57BL/6J mice. The number of 2-cell embryos is maximized when 10 IU PMSG/hCG is used in the embryo transplantation.