Pesticide residues, significantly hampering the overall environmental and human health, have become an increasingly severe issue. Thus, developing rapid, cost-effective, and sensitive tools for monitoring the pesticide residues in food and water is extremely important. Compared to the conventional and chromatographic techniques, enzyme inhibition-based biosensors conjugated with the fluorogenic probes provide effective alternative methods for detecting pesticide residues due to the inherent advantages including high selectivity and sensitivity, simple operation, and capability of providing in situ and real-time information. However, the detection efficiency of a single enzyme-targeted biosensor in practical samples is strongly impeded by the structural diversity of pesticides and their distinct targets. In this work, we developed a strategy of multienzyme-targeted fluorescent probe design and accordingly obtained a novel fluorescent probe (named as 3CP) for detecting the presence of wide variety of pesticides. The designed probe 3CP, targeting cholinesterases, carboxylesterases, and chymotrypsin simultaneously, yielded intense fluorescence in the solid state upon the enzyme-catalyzed hydrolysis. It showed excellent sensitivity against organophosphorus and carbamate pesticides, and the detection limit for dichlorvos achieved 1.14 pg/L. Moreover, it allowed for the diffusion-resistant in situ visualization of pesticides in live cells and zebrafish and the sensitive measurement of organophosphorus pesticides in fresh vegetables, demonstrating the promising potential for tracking the pesticide residues in environment and biological systems.