Organophosphorus pesticides (OPs) have been widely used in agricultural production. However, their potential harm to human health and the environment cannot be ignored. On-site detection of OPs in water is valuable for pollution source tracing and early warning of environmental pollution. We design an unmanned boat detection system equipped with a Raman spectrometer, which can achieve automated sampling and real-time detection of OPs in water. The unmanned boat detection system integrates automatic sampling, chemical reaction, spectral detection, and signal transmission modules. This method can specifically detect OPs in water by utilizing the inhibition effect of OPs on acetylcholinesterase activity. The minimum detection concentration is 700 nM for parathion-methyl. This method shows good anti-interference ability for other kinds of pesticides, demonstrating its applicability for rapid detection of OPs under complex water environmental conditions. Our strategy provides an efficient solution for real-time and flexible monitoring of OPs in water environments.