The identification of pollutants is crucial to protect water resources and ensure food safety. The available analytical methodologies allow reliable detection of organic pollutants such as pesticides; however, there is the need for faster, direct and continuous methodologies for real‐time monitoring of pesticides. Fluorescent‐based biosensors have been recently proposed as a valid alternative due to their advantage of being easy, cheap and specific. In this context, the aim of the present EU‐FORA fellowship programme was to develop and apply a fluorescence‐based biosensing device for the detection of organophosphate (OP) pesticides in water samples and drinkable food. The study was addressed using a mutant of the thermostable esterase‐2 from Alicyclobacillus acidocaldarius (EST2‐S35C) as a bioreceptor for OP pesticides. The use of EST2 involves some significant advantages including specificity and affinity towards OPs, and high stability over time in a different range of temperatures and pH. The protein was labelled to the fluorescent probe IAEDANS and fluorescence measurements of quenching in solution and in immobilised form were performed. The results showed good stability and sensitivity, reaching low limits of detection and quantification and a constant signal intensity over time. The addition of paraoxon quenched the fluorescence of the complex, reaching a plateau at 100 pmol paraoxon. The decrease of enzymatic activity of EST2‐S35C‐IAEDANS in the presence of paraoxon correlated the inhibition of the labelled enzyme with the decrease in fluorescence. The results from the application of the biosensor with real samples showed a decrease in fluorescence in surface water samples, contaminated by OPs. The use of the developed fluorescence‐based biosensor demonstrated its applicability for real samples monitoring and could ensure the production of large amounts of data in a short period of time which can be used to address environmental and food safety risk assessment.