Abstract: Umami substances play an important role in enhancing food flavor and ensuring human health, and biological sensing technology is an emerging method for detecting umami substances. Therefore, in this study, a highly sensitive detection method for monosodium glutamate (MSG) was developed based on competitive reactions that were mediated by polystyrene (PS) microspheres and MSG-BSA functionalized complexes. After washing and centrifugation, the polystyrene microspheres were decorated with MSG-BSA and competed with MSG to bind with magnetic beads (MB) loaded with MSG receptor T1R1 in the tested sample. The coupled complex was removed by magnetic separation, and the remaining polystyrene microspheres was counted by 15 s to achieve the detection of monosodium glutamate concentration. UV spectroscopy and Zeta potential analyzer were used to characterize the surface modification of polystyrene microspheres and magnetic beads. The characteristic absorption peaks of 250 nm and 270 nm were observed for the naked polystyrene microspheres labeled with MSG-BSA, while the characteristic absorption peaks of 600 nm and 210 nm were observed for T1R1-labeled magnetic beads. It was found that the particle size of both polystyrene microspheres and magnetic beads changed significantly, indicating the successful modification of their surface. To obtain the optimal detection performance, the concentration of polystyrene microspheres, magnetic beads, and incubation time were optimized. The optimal conditions for the experiment were identified as polystyrene microspheres diameter of 3 μm, magnetic beads diameter of 1 μm, concentration ratio of polystyrene microspheres and magnetic beads at 1:3, and incubation time of 10 min. Particle counter is highly sensitive and accurate in detecting the polystyrene microspheres state, and particles at different states present significant differences in resistance. This detection simplified the procedure and greatly improved the detection sensitivity. The response equation of the method to the tested monosodium glutamate solution was satisfactory with a fitting coefficient of R²=0.993 and a limit of detection of 5.17 pg/mL. Compared with other methods, this method yielded better linear range and detection limit.To verify the specificity of the detection system for taste substance detection, four interfering substances, namely citric acid, sucrose, benzalkonium chloride, and sodium chloride, were selected. The response value of the interference signal was negligible compared to that of monosodium glutamate, indicating the high specificity of this detection system.Three sets of monosodium glutamate (MSG) solutions with the same concentration in parallel configurations were tested, with each set tested 10 times. The intra-group and inter-group relative standard deviation (RSD) was calculated, demonstrating that this method provided good stability for MSG detection. Moreover, the sensitivity and applicability of the developed detection method for actual sample analysis were verified using commercially available crucian carp, carp, and grass carp as examples. After a series of pre-processing operations, the response values of different concentrations of supernatant from crucian carp, carp, and grass carp were determined. The results showed that as the concentration of the supernatant increased and within the linear response range of the method, the response value of the particle counter also increased, indicating that this method can be used to perceive the concentration of umami substances in fish samples. This study demonstrated that the developed method had good accuracy and sensitivity, showing great application potential in food preservation and quality evaluation.