A novel label-free fluorescence polarization (FP) assay utilizing bovine serum albumin nanoparticles (BSA NPs) in conjunction with manganese dioxide nanosheets (MnO2 NS) has been developed for the simultaneous detection of glucose, choline and lactate. This method eliminated the need for antibodies, aptamers, or labeled dyes. The BSA NPs exhibit a weak FP signal, while the MnO2 NS significantly amplificates the FP signal of the BSA NPs. The oxidizing properties of MnO2 NS result in a marked reduction of the amplified FP signal of the BSA NPs-MnO2 NS system with hydrogen peroxide (H2O2). Glucose, choline, and sodium lactate, which are prevalent food additives, can generate H2O2 through the action of their respective oxidases. Consequently, the BSA NPs-MnO2 NS system was employed as a label-free platform for the FP detection of glucose, choline, and sodium lactate within the concentration ranges of 0-200 μM, 0-800 μM, and 0-200 μM, respectively.