Herein, target-triggered magnetic nanoporous carbon-upconversion nanoparticles (Fe3O4@NPC-UCNPs) assembly as photoactivatable nanozymes were first used to build a novel fluorescence biosensor for sensitive detection of aflatoxin B1 (AFB1). In this study, the catalytic activity of Fe3O4@NPC was combined with heterostructure physical property of Fe3O4@NPC and optical property of UCNPs by immune recognition. The UCNPs absorbed near infrared light and transferred energy to Fe3O4@NPC, induced the separation of electron-hole pairs on the heterostructure, resulting in the generation of hydroxyl free radical (·OH). The Fe3O4@NPC-UCNPs assembly nanozymes could oxidize 2′,7′-dichlorodihydrofluorescein (DCFH) into 2′,7′-dichlorofluorescein with a high fluorescence intensity by ·OH and showed high affinity to DCFH (Km = 0.0799 mM). The designed fluorescence biosensor allowed quantitative detection as low as 0.56 pg/mL with a wide linear range of detection from 0.1 to 10 ng/mL. The light-activated Fe3O4@NPC-UCNPs assembly could take O2 as an oxidant to oxidate the substrate, avoiding the using of unstable H2O2 and endowing higher accuracy in biosensing applications.