HSSP is a 10 kD Zein gene modified with soybean codon. In the previous study, a single copy of transgenic soybean GSDH5 was screened from the genetically modified soybeans. In this study, the left boundary flanking sequence of the TDNA insertion site of the transformation soybean GSDH5 was obtained by chromosome walking method, and the left boundary flanking sequence was analyzed, the specific primers were designed, and the specific detection method of transformation soybean GSDH5 was established. Realtime PCR was used to detect the expression of foreign genes in different tissue parts of transgenic soybean (root, stem, leaf, flower and seed), RTPCR and Western blot were used to detect the genetic stability of the target gene at the transcriptional and translation levels. The contents of crude protein, sulfurcontaining amino acids and main agronomic characters in the transgenic soybean GSDH5 were determined and analyzed, which laid a foundation for cultivating new varieties of transgenic soybeans with high sulfur amino acids. The results showed that: (1) molecular identification showed that the exogenous gene HSSP and the screening marker gene Bar were successfully integrated into the soybean ‘Dongnong 50’ genome, and were integrated into the soybean genome in the form of single copy. (2) HSSP gene was successfully inserted into 52 873 883 bp of noncoding region of soybean genome 1. (3) HSSP gene was specifically expressed in the seeds of transgenic soybean GSDH5, and could be stably inherited and expressed in T₂-T₄ transgenic soybeans. (4) The range of crude protein content of ‘Dongnong 50’ was 41.53%-43.32%, and that of GSDH5 was 40.18%-43.03%. There was no significant difference between the two. Sulfur amino acids in GSDH5 seeds accounted for 1.35% of seed dry samples and 3.14% of seed protein, which increased significantly by 11% and 16%, respectively. (5) There were no significant differences between transgenic soybean GSDH5 plant and acceptor variety ‘Dongnong 50’ in pod number per plant, 100seed weight, plant height, pod forming habit, flower color, leaf shape and other aspects, which proved that the insertion of HSSP gene had no adverse effects on the growth and development of soybean plant. It is suggested that the transgenic soybean GSDH5 material has the potential to cultivate new soybean varieties with high sulfur amino acid content.