Abstract: Transcription factor HD-Zip family gene Gm01g04890 was introduced into cotyledonary nodes of sequenced soybean varieties Willimas 82 and small grain soybean varieties DN50 with Agrobacterium tumefaciens-mediated cotyledonary node transformation. Effect of seed germination time, Agrobacterium tumefaciens concentration, infection time, co-culture time, and acetosyringone (AS) concentration on overgrowing shoots of soybean cotyledonary node was studied. Genetic transformation system of soybean cotyledonary node was optimized. The transgenic plants were detected by glufosinate resistance screening and RT-PCR analysis. The data showed that the target gene was integrated into the soybean genome. The DN50 plants transformed with Gm01g04890 gene were obtained.