Lycopene content significantly increased in pulsed light-treated tomatoes. Proteomic analysis identified 1046 differentially expressed proteins (DEPs). Multi-omics analyses revealed that the gene expression, protein accumulation, and metabolite levels in the carotenoid biosynthesis pathway of tomatoes were coordinately upregulated after pulsed light treatment, with particularly significant activation of pathways related to the tricarboxylic acid (TCA) cycle, terpenoid backbone biosynthesis, and photosynthesis-antenna proteins. The findings indicated that the reprogramming of energy metabolism (including oxidative phosphorylation and carbon fixation pathways) and the synthesis of secondary metabolites (such as porphyrin metabolism and glutathione metabolism) played crucial roles in lycopene accumulation induced by pulsed light. These differential molecules were significantly enriched in pathways including terpenoid backbone biosynthesis, carotenoid biosynthesis, the tricarboxylic acid (TCA) cycle, and photosynthesis.