Abstract: In order to study different methods for determination of soybean pollen vitality, SuiNong4、JiLin47 and CK-P were studied, through single factor orthogonal test to sucrose ,agar, H3BO3 and temperature, filtered the culture medium for pollen germination , the difference of pollen viability between I2-KI, TTC staining and in vitro germination were compared. The results showed that the I2-KI staining method was easy to operate, because the method was more simple than the in vitro germination method, the determination result was accurate, the cost was low and the time was short,so it was used to test pollen vitality . TTC and I2-KI staining method was similar, there was no difference, but also can be used as a method to detect soybean pollen vitality. The method of vitro germination of pollen viability was related to many factors,and I2-KI and TTC staining were significantly different, and its preparations were complex and cumbersome, so I2-KI and TTC staining method can be used to determine soybean pollen viability.