Rapid and precise quantification of organophosphorus pesticides (OPPs) in environmental water bodies is crucial for evaluating ecological risks and safeguarding human health. Traditional instrumental methods are complex, time-consuming, and expensive, while enzyme-based biosensors suffer from instability and require a constant supply of substrates. Hence, there is an urgent need for a fast, simple, and sensitive biosensor for OPPs. In this study, we developed a novel non-enzymatic biosensor for the detection of methyl parathion (MP) by employing the bioluminescence resonance energy transfer (BRET) Q-body strategy. Optimizing the spacer arm and screening fluorescent dyes identified the R6G BRET MP Q-body sensor with the best performance. Key parameters affecting the sensor's detection performance were optimized by using single-factor experiments. Under optimal conditions, the detection exhibited a detection limit of 5.09 ng·mL−1 and a linear range of 16.21–848.81 ng·mL−1. The sensor's accuracy was validated using standard recovery experiments, yielding a recovery rate of 84.47 %–102.08 % with a standard deviation of 1.93 %–9.25 %. The detection results of actual water samples demonstrate that this fast, simple, and highly sensitive BRET Q-body sensor holds great promise for practical water quality monitoring.