Fructose-1,6-bisphosphate (FBP), a cellular endogenous sugar metabolite in the glycolytic pathway, has recently been reported to act as a signaling molecule to regulate various cellular events through the engagement of important proteins. Though tremendous progress has been made in identifying specific FBP-protein interactions, the comprehensive identification of FBP-interacting proteins and their regulatory mechanisms remains largely unexplored. Here, we describe a concise synthetic approach for the scalable preparation of a photoaffinity FBP probe that enables the quantitative chemoproteomic profiling of FBP-protein interactions based on photoaffinity labeling (PAL) directly in living cells. Using such a protocol, we captured known FBP targets including PKM2 and MDH2. Furthermore, among unknown FBP-interacting proteins, we identified a mitochondrial metabolic enzyme aldehyde dehydrogenase 2 (ALDH2), against which FBP showed inhibitory activity and resulted in cellular ROS upregulation accompanied by mitochondrial fragmentation. Our findings disclosed a new mode of glucose signaling mediating by the FBP-ALDH2-ROS axis.