Abstract: Through the investigation and analysis of underwater oil spill monitoring technology and the 3D fluorescence spectrum analysis of actual oil samples, a high sensitivity and high precision underwater oil spill fluorescence sensor is developed in this work. According to the comprehensive consideration of the wavelength band gap of the filter, the wavelength band of the sample response, and the wavelength and performance of the light emitting diode (LED) under the existing production process, the two excitation wavelengths were selected at 275 nm (Channel 1) and 365 nm (Channel 2). The simulation results of the two optical paths show that the illumination or collection effect of the sensor at its target distance is in line with the expectation. Two kinds of crude oil samples from different mining areas in the South Sea of China were tested with this sensor. The detection limits of crude oil Sample A and Sample B were 2.8 μg/L and 3.5 μg/L respectively in Channel 1 of the dual-channel oil spill fluorescence detector. The detection limits for Sample A and Sample B in Channel 2 are 8.6 μg/L and 1.8 μg/L respectively. The detection limits are better than common commercial instruments of the same type. The low detection limit allows the sensor to continuously detect low oil concentration signals in the ocean. In summary, the sensor has a good application prospect in the monitoring of oil spill in the sea.