In this study, we developed a highly sensitive glutamate biosensor based on Pt nanoparticles (PtNP) decorated two-dimensional nanomaterial MXene-Ti3C2Tx for sensitive analysis of monosodium glutamate (MSG) in food samples. The morphology of PtNP@MXene-Ti3C2Tx was characterized by SEM, which confirmed the successful synthesis of the nanocomposite. The electrochemical experiments were carried out after optimizing working conditions, and a linear relationship was established between the current response and MSG content. The biosensor exhibited a good linear relationship with a correlation coefficient (R2) of 0.9948 in a range of 10–110 μmol/L, and it also had excellent sensitivity (1.5906 nA/(μmol/L)) and a low detection limit (0.45 μmol/L). The biosensor exhibited high selectivity towards glutamate, and it could hold over 70% of its initial sensitivity for 28 days when stored under optimal conditions. The analytical recovery of added MSG (50 and 100 μmol/L) in the vegetable soup was 87.04 and 106.81 g/100 g, respectively. The biosensor was also used successfully to test MSG content in soy sauce, stock cube, and mushroom seasoning. Hence, the constructed glutamate biosensor would be a suitable method for rapid detection of MSG in foodstuffs.