The invention provides a novel synchronous detection method of soil rhizobium japonicum separation and nitrogen fixation activity. The method comprises the following steps: selecting soybean used for production, washing the soybean with sterile water and airing the soybean; repeatedly disinfecting the soybean with 0.1% mercury dichloride for more than 3 times. Putting vermiculite into a plastic nutrition pot whose diameter is 10 cm, sterilizing the nutrition pot in a high-temperature high-pressure steam sterilizing pot; sticking the disinfected soybean seed respectively to soil and rhizobium; using the disinfected seed as reference, seeding the soybean and the reference seed in the nutrition pot at the same time, and placing the nutrition pot into an aseptic bag which has a ventilation sterilization cover; putting the aseptic bag into a light cultivating box for cultivation; periodically watering the soybean seed with Hogland nutrient solution until the fourth compound leaf of the soybean completely spread out; if the reference seed has no root nodule, detecting the rhizobium dinitrogenase activity of the soil and the soybean with root nodule; meanwhile, separating rhizobium from the root nodule and implementing 16srDNA identification, comparing with the dinitrogenase activity of the root nodule cultivated from the rhizobium, reserving the high nitrogen fixation activity rhizobium japonicum obtained from separation.