Fluorescence has unique luminescence characteristics. The combination of excitation light and emission light can greatly reduce the interference of background signals and greatly improve the sensitivity of the detection system. Many scholars at home and abroad obtain the optimal excitation and emission wavelengths of the analyte based on the principle of photoluminescence of fluorescent substances, and have developed miniaturized, low-cost dedicated instruments. However, the current portable instruments which focus on detecting pesticide residues in the solution system cannot realize directly detection pesticide residues on the surface of vegetable leaves. In this study, a feasible model for pesticide residues and fluorescence intensity on the leaf surface was proposed, and a portable detection instrument for pesticide residues on the leaf surface was designed by optimizing the light path structure to suppress the interference of stray light on the leaf surface. Firstly, the best excitation wavelength and best emission wavelength of acetamiprid pesticide in standard solution and three plant leaves were determined as 350 and 500 nm, respectively. An LED with a central wavelength of 350 nm and a maximum drive current of 100 mA was selected as the excitation light source, and a photodiode with a peak response range from 480 to 580 nm was used as the photoelectric detection device. A filter with a center wavelength of 500 nm was used to only let through the emission wavelength of the acetamiprid pesticide pass to reduce the interference of the secondary spectrum. Then the optimal parameters of the optical path are determined. The fluorescence experiments results showed that the fluorescence intensity excited by acetamiprid pesticide on the leaf surface was the highest when the light Angle was 45°. By calculating the optical path size, it was found that the illumination of the blade was the largest when the direct distance from the LED laser source to the blade was 4.89 cm and the vertical distance was 3.46 cm. In addition, in view of the diffuse reflection problem of the light source illuminating the surface of the blade, a diffuse reflection device was designed to achieve the maximum absorption of light energy. The control circuit, driving circuit and detection circuit were designed according to the requirement of weak fluorescence signal detection. A signal detection system was designed with STM32 chip as the main control chip to collect the voltage signal of the detection circuit, and the pesticide residue value was calculated according to the working curve of pesticide residue value. The PWM wave was output to modulate the LED light source and the difference between the frequency of detecting light and the frequency of ambient light can suppress the interference of ambient light to the light source. Then, SPI and I2C communication protocols were used to communicate with AD acquisition chip and OLED display screen to realize real-time detection of pesticide residues and real-time display of pesticide residues. Finally, the calibration equation was established and a portable detector was designed to detect pesticide residues. The measuring instrument was calibrated and tested.The determination coefficient of the calibration equation reached 0.875, and the root mean square error is 0.405 mg/L. The portable fluorescence spectrometer designed in this study can quickly, accurately and non-destructively detect pesticide residues on the surface of leaves, which provided a reference for the development of a more universal portable detection instrument.