Barley is the fourth largest cereal crop in the world and is widely used in the fields of grain, brewing, feed, food and medicine. Caryopsis is an important organ for nutrient storage in barley, and the nutrient substances accumulated in caryopsis are mainly starch and protein. The development and substance accumulation of caryopsis directly determine the yield and quality of barley. As one of the important abiotic stress factors, drought is a common problem in global food production, which seriously restricts the production of barley. However, the mechanism underlying starch and protein accumulation in barley caryopsis under drought stress remains unclear. In this study, Suluomai 1 was selected as material and subjected to drought treatment from flowering to caryopsis maturity stage. The characteristics of substance accumulation and hormone changes in barley caryopsis were studied by resin slicing, microscopic observation and physiological determination. Drought stress reduced starch accumulation but increased protein accumulation in the endosperm of barley caryopsis. Meanwhile, auxin and gibberellin contents in barley caryopsis decreased, zeatin riboside and abscisic acid content increased under drought stress. Moreover, the gene expression characteristics in barley caryopsis were analyzed by RNA-seq, and the possible mechanism of substance accumulation in caryopsis under drought stress was revealed from the perspective of hormone regulation. A total of 2,642 differentially expressed genes were screened, of which 1,192 were up-regulated and 1,450 were down-regulated. Among those differentially expressed genes, 93 hormone-related genes were selected. These genes played a certain role in regulating substance accumulation of barley caryopsis in response to drought by affecting hormone synthesis and signal transduction. The research results can provide theoretical basis for drought-tolerant breeding and quality improvement of barley.