Catalytic redox reactions have been employed to enhance colorimetric biodetection signals in point-of-care diagnostic tests, while their time-sensitive visual readouts may increase the risk of false results. To address this issue, we developed a dual photocatalyst signal amplification strategy that can be controlled by a fixed light dose, achieving time-independent colorimetric biodetection in paper-based tests. In this method, target-associated methylene blue (MB + ) photocatalytically amplifies the concentration of eosin Y by oxidizing deactivated eosin Y (EYH 3− ) under red light, followed by photopolymerization with eosin Y autocatalysis under green light to generate visible hydrogels. Using the insights from mechanistic studies on MB +sensitized photo-oxidation of EYH 3− , we improved the photocatalytic efficiency of MB + by suppressing its degradation. Lastly, we characterized 100-to 500-fold enhancement in sensitivity obtained from MB + -specific eosin Y amplification, highlighting the advantages of using dual photocatalyst signal amplification.