AbstractTrimethylamine monooxygenase (Tmm, EC‐1.14.13.148) belongs to the family of flavin‐containing monooxygenases that oxidize trimethylamine into trimethylamine‐N‐oxide (TMAO). Conventional methods for assaying Tmm are accurate over a narrow range of substrate/product concentrations. Here we report a TMAO‐specific enzymatic assay for Tmm using polyallylamine hydrochloride (PAHCl)‐capped MnO2 nanoparticles (PAHCl@MnO2). We achieved TMAO specificity using iodoacetonitrile to remove interfering trimethylamine. The change in the concentration of TMAO is measured by observing the difference in the absorbance of 3,3′,5,5′‐tetramethylbenzidine (TMB) at 650 nm. The assay is tolerant to several interfering metal ions and other compounds. This method is more accessible and reliable than currently known methods. The limit of detection (LOD) and limit of quantitation (LOQ) are 1 μM and 10 μM, respectively, for direct TMAO measurement.