To set a systematic study of the Sorghum cystatins (SbCys) gene family, a genome-wide analysis of the SbCys family genes was performed by bioinformatics-based methods. In total, 18 SbCys genes were identified in Sorghum, which were distributed unevenly on chromosomes, and two genes were involved in a tandem duplication event. All SbCys genes had similar exon/intron structure and motifs, indicating their high evolutionary conservation. Transcriptome analysis showed that 16 SbCys genes were expressed in different tissues, and most genes displayed higher expression levels in reproductive tissues than in vegetative tissues, indicating that the SbCys genes participated in the regulation of seed formation. Furthermore, the expression profiles of the SbCys genes revealed that seven cystatin family genes were induced during Bipolaris sorghicola infection and only two genes were responsive to aphid infestation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that 17 SbCys genes were induced by one or two abiotic stresses (dehydration, salt, and ABA stresses). The interaction network indicated that SbCys proteins were associated with several biological processes, including seed development and stress responses. Notably, the expression of SbCys4 was up-regulated under biotic and abiotic stresses, suggesting its potential roles in mediating the responses of Sorghum to adverse environmental impact. Our results provide new insights into the structural and functional characteristics of the SbCys gene family, which lay the foundation for better understanding the roles and regulatory mechanism of Sorghum cystatins in seed development and responses to different stress conditions.