ABSTRACT Light is one of the essential environmental factors that significantly influences the biosynthesis of secondary metabolites in plants. To explicate their effect, exposure of monochromatic LED light spectra (yellow, blue, green, red, and white) on the accumulation of capsaicinoids, phenolic compounds, antioxidant capacity, and regulation of transcriptional changes in capsaicinoids biosynthesis genes at 24, 48, and 72 h in chili callus cultures was investigated. Blue light significantly increased capsaicin and dihydrocapsaicin contents by 41.43‐fold and 4.36‐fold, respectively, compared to white light. Pearson correlation analysis confirmed a strong positive correlation between total phenolics, total flavonoids, and antioxidant capacity ( r > 0.85). Gene expression analysis revealed that the biosynthetic genes ( pAMT, KAS, ACS, ACL, CL, C4H , and CS ) were upregulated, and their associated transcription factors—MYB, ERF, and JERF—showed varied expression, indicating their putative regulatory role in capsaicinoid biosynthesis under different monochromatic LED conditions. Our findings demonstrate that the targeted monochromatic LED lights can enhance bioactive compound production in C. annuum callus cultures, providing a favorable strategy for feasible production of the industrially important compound “capsaicin” on a large scale for pharmaceutical and food applications.