The UNUSUAL FLORAL ORGANS (UFO) gene is an essential regulatory factor of class B genes and plays a vital role in the process of inflorescence primordial and flower primordial development.The role of UFO genes in soybean was investigated to better understand the development of floral organs through gene cloning, expression analysis, and gene knockout. There are two copies of UFO genes in soybean and in situ hybridization, which have demonstrated similar expression patterns of the GmUFO1 and GmUFO2 genes in the flower primordium. The phenotypic observation of GmUFO1 knockout mutant lines (Gmufo1) showed an obvious alteration in the floral organ number and shape and mosaic organ formation. By contrast, GmUFO2 knockout mutant lines (Gmufo2) showed no obvious difference in the floral organs. However, the GmUFO1 and GmUFO2 double knockout lines (Gmufo1ufo2) showed more mosaic organs than the Gmufo1 lines, in addition to the alteration in the organ number and shape. Gene expression analysis also showed differences in the expression of major ABC function genes in the knockout lines. Based on the phenotypic and expression analysis, our results suggest the major role of GmUFO1 in the regulation of flower organ formation in soybeans and that GmUFO2 does not have any direct effect but might have an interaction role with GmUFO1 in the regulation of flower development. In conclusion, the present study identified UFO genes in soybean and improved our understanding of floral development, which could be useful for flower designs in hybrid soybean breeding.