<p>Glucosinolates are a crucial class of secondary metabolites in cruciferous plants, widely recognized for their significant biological activities. This study aimed to investigate the effect of combined selenium and sulfur applications on glucosinolate biosynthesis and the expression of key regulatory genes in Chinese cabbage sprouts (<i>Brassica rapa</i> L. subsp. <i>pekinensis</i>). The experiment was conducted using the cultivar ‘Degao 536’ as the experimental material. Treatments included ZnSO₄, Na₂SeO₃, and their combined application, with the aim of evaluating their respective effects on glucosinolate accumulation and gene expression. Results demonstrated that ZnSO₄ treatment significantly upregulated the expression of genes involved in glucosinolate biosynthesis, including structural genes <i>BrCYP79F1, BrMAM1, BrCAT4, BrCYP79B2, BrCYP79B3</i>, and <i>BrCYP83B1,</i> as well as the regulatory genes <i>BrMYB28,</i> and<i> BrMYB51</i>. In contrast, Na₂SeO₃ treatment markedly suppressed the expression of these genes. However, the combined application of ZnSO₄ and Na₂SeO₃ effectively alleviated this suppression. Furthermore, ZnSO₄ treatment significantly increased the accumulation of aliphatic, indolic, aromatic glucosinolate groups, and the total glucosinolate content. Conversely, Na₂SeO₃ treatment led to a significant reduction in the levels of aliphatic, indolic glucosinolates, along with total glucosinolate. Notably, the combined application of selenium and sulfur treatment mitigated the inhibitory effects of selenium alone on glucosinolate synthesis.</p>