Promoters play a crucial role in controlling the spatial and temporal expression of genes at transcriptional levels in the process of higher plant growth and development. The spatial, efficient, and correct regulation of exogenous genes expression, as desired, is the key point in plant genetic engineering research. Constitutive promoters widely used in plant genetic transformation are limited because, sometimes, they may cause potential negative effects. This issue can be solved, to a certain extent, by using tissue-specific promoters. Compared with constitutive promoters, a few tissue-specific promoters have been isolated and applied. In this study, based on the transcriptome data, a total of 288 tissue-specific genes were collected, expressed in seven tissues, including the leaves, stems, flowers, pods, seeds, roots, and nodules of soybean (Glycine max). KEGG pathway enrichment analysis was carried out, and 52 metabolites were annotated. A total of 12 tissue-specific genes were selected via the transcription expression level and validated through real-time quantitative PCR, of which 10 genes showed tissue-specific expression. The 3-kb 5′ upstream regions of ten genes were obtained as putative promoters. Further analysis showed that all the 10 promoters contained many tissue-specificcis-elements. These results demonstrate that high-throughput transcriptional data can be used as effective tools, providing a guide for high-throughput novel tissue-specific promoter discovery.