Immense use of an insecticide such as malathion has initiated a hindrance to the environment and has increased possible risk to human health via milk and milk products. The presence of insecticide residues in milk products is an alarming signal to set up a string monitoring system. Since their risk to human health, the determination of organophosphate pesticide levels in dairy products is essential. This study shows biosensors' development for the detection of residues of the pesticides generally used in agriculture to increase productivity, which has high toxicity and selective action, a significant part of which falls into crop and livestock products. The test system described in this paper is based on the immobilization of acetylcholinesterase on glass rods by colorimetry as a detection technique. The application of the biosensor to the analysis of the real sample confirmed its reliability. The developed biosensor test system showed excellent analytical performance with limits to detecting the concentration range of residual amounts of the acetylcholinesterase enzyme inhibitor - malathion insecticide (up to 0.089 mg/kg), along with acceptable reproducibility and stability. Distinct advantages of enzyme-based biosensors are high sensitivity, convenience, quick response, cost-effectiveness, portability, and a possibility to use for environmental detection purposes. The biosensor can be used for the analysis of organophosphorus pesticides with anticholinesterase action. It can be concluded that the benefits of using immobilized enzymes are significant since it will bring advantages for the food, pharmaceutical, and medical device industries.