MicroRNAs (miRNAs) widely participate in plant growth and development. ThemiR396family, one of the most conserved miRNA families, remains poorly understood in sorghum. To reveal the evolution and expression pattern ofSbi-miR396gene family in sorghum, bioinformatics analysis and target gene prediction were performed on the sequences of theSbi-miR396gene family members. The results showed that fiveSbi-miR396members, located on chromosomes 4, 6, and 10, were identified at the whole-genome level. The secondary structure analysis showed that the precursor sequences of all fiveSbi-miR396potentially form a stable secondary stem–loop structure, and the mature miRNA sequences were generated on the 5′ arm of the precursors. Sequence analysis identified the mature sequences of the fivesbi-miR396genes were high identity, with differences only at the 1st, 9th and 21st bases at the 5’ end. Phylogenetic analysis revealed thatSbi-miR396a,Sbi-miR396b, andSbi-miR396cwere clustered into Group I, andSbi-miR396dandSbi-miR396ewere clustered into Group II, and all fivesbi-miR396genes were closely related to those of maize and foxtail millet. Expression analysis of different tissue found thatSbi-miR396d/eandSbi-miR396a/b/cwere preferentially and barely expressed, respectively, in leaves, flowers, and panicles. Target gene prediction indicates that the growth-regulating factor family members (SbiGRF1/2/3/4/5/6/7/8/10) were target genes ofSbi-miR396d/e. Thus,Sbi-miR396d/emay affect the growth and development of sorghum by targetingSbiGRFs. In addition, expression analysis of different tissues and developmental stages found that allSbi-miR396target genes,SbiGRFs, were barely expressed in leaves, root and shoot, but were predominantly expressed in inflorescence and seed development stage, especiallySbiGRF1/5/8. Therefore, inhibition the expression ofsbi-miR396d/e may increase the expression ofSbiGRF1/5/8, thereby affecting floral organ and seed development in sorghum. These findings provide the basis for studying the expression of theSbi-mir396family members and the function of their target genes.