The invention relates to a method for determining the number of soybean cotyledon cells. The method can solve the problems that the existing method for determining the number of the soybean cotyledoncells has the shortcomings of cumbersome steps, complicated operation and inaccurate determination result. The method for determining the number of the soybean cotyledon cells is carried out accordingto the following steps: 1, drying and grinding; 2, chromic acid dissolution; 3, sampling and determination; and 4, calculating, thereby obtaining the number of the soybean cotyledon cells. The methodfor determining the number of the soybean cotyledon cells has the advantages of accurate determination of the number of the cotyledon cells, simple determination steps, convenient operation and moreprecise determination result. The method for determining the number of the soybean cotyledon cells provides an important technical support for correct evaluation of the relationship between the numberof the cotyledon cells of different grain weight genotypes of soybeans and filling rate, accumulation of dry matters and dynamics of hormones.