BACKGROUNDIncreasing food safety awareness of consumers promotes the development of rapid and sensitive detection techniques for pesticide residues. In this study, a new type of rapid detection card for organophosphorus and carbamate pesticide residues was developed by electrospinning. The card involved enzyme fiber mat (EFM) and substrate fiber mat (SFM) which were prepared by mixing poly(vinyl alcohol) with acetylcholinesterase (AChE) and indolyl acetate (IA), respectively.RESULTSThe mean diameter of fibers was 240 ± 53 nm for EFM and 387 ± 84 nm for SFM. Results of Fourier transform infrared and X‐ray photoelectron spectroscopies confirmed that AChE and IA were successfully encapsulated into the fibers. The minimum concentrations of AChE and IA for effective detection were 1 and 3 mg mL−1, respectively, and the optimal detection time was 15 min. The limits of detection for this card were 0.5 mg L−1 for omethoate, 1.5 mg L−1 for malathion, 0.1 mg L−1 for carbaryl and 0.02 mg L−1 for carbofuran. The detection card exhibited good storage stability and its activity could be maintained when stored at room temperature for at least 4 months. Additionally, the EFM can be reused three times.CONCLUSIONSThe detection card obtained here was superior to a commercial card in detecting pesticide residues in real food samples. Hence, this electrospun detection card has potential for simple, rapid and sensitive analysis of pesticide residues. © 2020 Society of Chemical Industry