Abstract The simultaneous determination of pesticides chlorpyrifos, imazalil and thiabendazole in drinking water and wine samples with a biosensor based on White Light Reflectance Spectroscopy is presented. A competitive immunoassay format was implemented followed by reaction of immunoadsorbed anti-analyte specific antibody molecules with a secondary antibody in order to enhance the signal and reduce the analysis time. The whole analysis was completed in less than 10 min and the limits of quantification achieved with calibrators prepared in buffer were 60 pg/mL for chlorpyrifos and imazalil, and 80 pg/mL for thiabendazole. Bottled water samples were analysed without any treatment, whereas white and red wine samples were analysed after 10-times dilution with assay buffer. This way pesticide concentrations as low as 0.6 ng/mL for chlorpyrifos and imazalil, and 0.8 ng/mL for thiabendazole could be detected in wines. The measurements were repeatable with intra- and inter-assay CVs values ranging from 4.9 to 8.2% and from 7.3 to 9.7%, respectively, for all assays. The accuracy of the measurements was evaluated through recovery experiments using spiked water and wine samples. Percent recovery values ranged from 86 to 116%, demonstrating the accuracy of the measurements performed with the developed biosensor. In addition, 5 wines spiked with different concentrations of the three pesticides were analysed both with the developed biosensor and a validated LC–MS/MS method. The good agreement of the results obtained with the two methods is a further testimony of the developed method accuracy and a good starting point to demonstrate its Point-of-Need application capabilities in the food analysis field.