Light quality exerts a vital influence on the accumulation of secondary metabolites in medicinal plants. Atropa belladonna L. serves as a primary source plant of tropane alkaloids (TAs). Nevertheless, in agricultural production, its application is restricted due to the relatively low content of alkaloids. This study explored the impacts of red, yellow, blue, and white light on the growth of A. belladonna and the biosynthesis of TAs. Through phenotypic and physiological analyses, it was found that red-light had the most significant effect on A. belladonna. Red-light remarkably increased the content of TAs, enlarged the leaf area, extended the stomatal length, and elevated the ammonium nitrogen level. It also enhanced the activities of ornithine decarboxylase, nitrate reductase, and glutamine synthetase, which are essential for nitrogen assimilation. Transcriptomic analysis identified GDHA, At2g42690, and PAO5 as key genes with upregulated expression in the putrescine biosynthesis pathway, where putrescine is an important precursor of TAs. Metabolomic data confirmed that the levels of scopolamine, hyoscyamine, and their precursors increased under red-light. Subsequent qPCR verification under red/white light treatments consistently showed the upregulation of these genes, further confirming their roles in the synthesis of TAs. Moreover, red-light activated photosynthesis-related genes and transcription factors, indicating a coordinated regulatory relationship between light signal transduction and metabolic pathways. This study has preliminarily elucidated the potential mechanism by which red-light promotes the accumulation of TAs through enhancing nitrogen metabolism and precursor synthesis, providing a theoretical basis for improving the quality of A. belladonna and optimizing agricultural production.