The FvC5SD gene from Flammulina velutipes was transformed into soybean cultivar Shennong 9 by Agrobacterium mediated genetic transformation of soybean cotyledon nodes. The transgenic soybean line FvC5SD-L05 showed good drought tolerance and agronomic performance. To clarify the characteristics of the transgenic soybean FvC5SD-L05 material meeting its biosafety evaluation, we analyzed the copy number of FvC5SD gene in T4 generation plant, by Southern hybridization and identified the position and direction of the insert in soybean genome. Meanwhile, by PCR amplification of the flanking sequences of exogenous T-DNA, we established a specific qualitative PCR detection method for FvC5SD-L05. This method represents specificity with high sensitivity, e.g. being able to detect transgenic fragment in the template with FvC5SD-L05 genomic DNA content of 0.1%. The specific detection of FvC5SD-L05 provided technical basis to identify the transgenic soybeans with the target gene.