In this study, the optimal production of indirubin and conversion of indigo extracted from waste blue jeans into indirubin, a precursor for anticancer drugs, are presented. Indirubin shares the same biosynthetic pathway as indigo, a dye commonly used in the production of jeans, but is synthesized through asymmetric dimerization. During this process, 2-oxoindole and isatin function as key precursors. Various conditions, including the T4MO enzyme, surfactants, bioconversion reactions, and the extraction solvent, were optimized to achieve efficient indirubin production. A major challenge in this process is the simultaneous production of both indigo and indirubin. Considering that indigo has lower photostability compared to indirubin, and its photodegradation yields isatin-a precursor for indirubin biosynthesis-the possibility of converting indigo to indirubin via photodecomposition was explored. This innovative approach was successfully validated. As a result, a yield of 5.95 mg of indirubin per pair of jeans was achieved through a bioconversion process, in which indigo was extracted from waste jeans and subsequently converted to indirubin following light-induced decomposition. This research demonstrates the potential for recycling waste jeans through an upcycling process, turning them into valuable precursors for anticancer drug development.