Increased consumer awareness on food safety has spurred the development of detection techniques for pesticide residues. In this study, a rapid detection card on the basis of enzyme action was developed for the visual detection of pesticides, in which the thermally crosslinked and surface-decorated polyvinyl alcohol/citric acid nanofiber mat (PCNM) was employed as a novel immobilization matrix for acetylcholinesterase (AChE). The PCNM, crosslinked at 130 °C for 50 min, exhibited appropriate microstructure and water stability, making it suitable for AChE immobilization. The activation of carboxyl groups by surface decoration resulted in a 2.5-fold increase in enzyme loading capacity. Through parameter optimization, the detection limits for phoxim and methomyl were determined to be 0.007 mg/L and 0.10 mg/L, respectively. The detection card exhibited superior sensitivity and a reduced detection time (11 min) when compared to a commercially available pesticide detection card. Furthermore, the detection results of pesticide residues in fruit and vegetable samples confirmed its feasibility and superiority over commercial alternatives, suggesting its great potential for practical application in the on-site detection of pesticide residues.