Low temperatures are the main abiotic factor affecting wheat growth. Brassinolide is a novel plant hormone that can improve the cold resistance of plants; however, the molecular mechanism of brassinolide in winter wheat at low temperatures remains unclear. In this study, winter wheat Dongnong dongmai 1 was sprayed with 0.01, 0.1, or 1.0 mg·L-1 brassinolide (BR) at the three-leaf stage, and tillering nodes were sampled at different temperatures (5, -10 and -25 °C), and then physiological indexes were determined and the transcriptome was sequenced. The results showed that the optimum concentration of brassinolide for cold resistance is 0.1 mg·L-1. A total of 15 302 (8198 upregulated and 7104 downregulated) differentially expressed genes (DEGs) were identified in the B1 vs D1 comparison (B1 represents 5 °C 0.1 mg·L-1 BR treatment, D1 represents 5 °C control); 3386 (1930 upregulated and 1456 downregulated) differentially expressed genes (DEGs) were identified in the B2 vs D2 comparison (B2 represents -10 °C 0.1 mg·L-1 BR treatment, D2 represents -10 °C control); and 2684 (2102 upregulated and 582 downregulated) differentially expressed genes (DEGs) were identified in the B3 vs D3 comparison (B3 represents -25 °C 0.1 mg·L-1 BR treatment, D3 represents -25 °C control). Further studies showed that these DEGs were mainly involved in carbon fixation in photosynthetic organs, photosynthesis and plant-pathogen interactions, all of which were related to stress and energy metabolism. This indicates that brassinolide can produce substances that improve cold resistance in wheat seedlings. This study provides a theoretical basis for further research on the improvement of cold resistance in winter wheat by brassinolide.