Prodigiosin is a tripyrrole-based microbial pigment that exhibits diverse biological activities, including antioxidant, antimicrobial, and anticancer properties. Moreover, it possesses excellent color fastness and dye uptake, making it a promising candidate in medicine, cosmetics and dye industries. In this study, the high-prodigiosin-producing mutant Serratia marcescens strain GEN-M54 was obtained through sequential physical and chemical mutagenesis. The effect of light on the prodigiosin production was investigated, and a two-step sequential light fermentation strategy was developed to promote the prodigiosin accumulation. Additionally, gene transcription analysis was conducted to elucidate changes in the metabolic flux of prodigiosin precursor synthesis under red-light stress conditions. Finally, a prodigiosin production of 6623.1 mg/L and a content of 471.4 mg/g were achieved through the two-step fermentation strategy, representing an increase of 77.1% and 46.8%, respectively. Overall, this study provides an efficient microbial platform and an efficient fermentation strategy for future prodigiosin production.