This paper describes the prototype of an on-site High Sensitivity Early Warning Monitoring System, using Photo-Induced Fluorescence, for pesticide monitoring in natural waters (HSEWPIF). To obtain a high sensitivity, the prototype was designed with four main features. Four UV LEDs are used to excite the photoproducts at different wavelengths and select the most efficient one. Two UV LEDs are used simultaneously at each wavelength to increase the excitation power and then the fluorescence emission of the photoproducts. High-pass filters are used to avoid the saturation of the spectrophotometer and to increase the signal-to-noise ratio. The HSEWPIF prototype also employs UV absorption to detect any occasional increase of suspended and dissolved organic matter, which could disrupt the fluorescence measurement. The conception of this new experimental set-up is explained and described, then analytical applications are carried out online for the determination of fipronil and monolinuron. We obtained a linear calibration range from 0 to 3 μg mL−1 with limits of detection of 1.24 ng mL−1 for fipronil and 0.32 ng mL−1 for monolinuron. A mean recovery of 99.2% for fipronil and 100.9% for monolinuron show that the method is accurate, moreover a standard deviation of 1.96% for fipronil and 2.49% for monolinuron show that the method is repeatable. Compared to other methods for the determination of pesticides by photo-induced fluorescence, the HSEWPIF prototype has good sensitivity with better limits of detection, and good analytical performances. These results show that HSEWPIF can be used for monitoring pesticide in natural waters to protect industrial facilities against accidental contamination.