We developed an enzymatic activity visualization platform that uses active intracellular enzymes as molecular baits to capture specific substrate probes. This platform links enzyme-mediated biochemical activity to their in situ localization, isolation, phylogenetic identification, and functional validation. Combined with a highly specific alkyne-azide cycloaddition reaction, these probes enable fluorescent visualization of strains harboring isozymes in human gut microbiota under anaerobic conditions. By performing metabolic capacity tests, fluorescence imaging, and proteomic analyses on four positive and three negative reference strains, we validated that our probes exhibit selectivity at the bacterial cell level. Using the platform, we successfully identified functional guilds involved in the reduction of sennoside A, a widely used laxative prodrug activated by gut bacteria.