Despite the tremendous achievements of existing point-of-care testing (POCT) techniques, accurate on-site detection of pesticide remains a thorny issue. Herein, the concept of multi-enzyme cascade system-based hydrogel kit integrated with a smartphone detector for in-field screening of pesticide was proposed. In this work, we constructed a target-responsive hydrogel (TRhg)-based kit by embedding MnO₂nanoflakes (NFs) into sodium alginate hydrogel. Based on remarkable mimic oxidase activity, MnO₂nanozyme induced color reaction via introducing the sensing probe 3,3′,5,5′-tetramethylbenzidine (TMB). Acetylcholinesterase (AChE) and choline oxidase (ChO) catalyzed the hydrolysis of acetylcholine (ACh) to produce H₂O₂, which triggered MnO₂NFs decomposition, further blocked the oxidation of TMB. Pesticide (paraoxon) as inhibitor of AChE suppressed the generation of H₂O₂, which reduced the decomposition of MnO₂NFs, resulting in the color response of kit. Interestingly, the images of kit that captured through smartphone could be analyzed via self-made application program. The benchtop-size biosensor was capable of screening pesticide within 65 min in a sensitive manner (detection limit of 0.5 ng mL-1). More importantly, the obtained results were consistent with those of traditional laboratory microplate reader. Such a miniaturized sensor exhibited high reliability, excellent long-term stability and ease of operation for real-time measurement of pesticide.