Bioassay is a traditional and classical method for analyzing the herbicides tolerance variation in plants, which has been widely adopted in determining the tolerance of crops, e.g. soybean, barnyard grass, and cotton, to herbicides, such as glyphosate and,chlorimuron-ethyl. However, the dicamba bioassay method and the tolerance variation of dicamba in different soybean cultivars remained uncharacterized. In this study, inhibition rate of hypocotyl elongation was used as the major index to evaluate the phenotypic variance of germinated soybean treated with dicamba. The dicamba bioassay was established by using 300 μg/L dicamba as threshold of screening concentration, and combining with the regression equation analysis and median inhibitory concentration (IC50) analysis.This method was used to identify 35 soybean varieties derived from micro-core germplasm, namely Dahuangdou-1 and Shifangluosidou, showed relatively higher tolerance to dicamba. Our study provided reference and theoretical basis for the cultivation of bicamba-resistant transgenic soybean receptor using transgenic technique and traditional crossing breeding or the selection of recurrent parent, and as well as the quick phenotypic determination of transgenic soybean with resistance to bicamba.