Glutathione (GSH) is an essential natural antioxidant, and plays a crucial role in the prevention of various diseases. Herein, a nitrogen-doped polydopamine manganese nanoparticle (N-PDA@Mn) was prepared and used as nanozyme sensor for the rapid and colorimetric detection of GSH. N-PDA@Mn demonstrated excellent oxidase-like activity (Km = 0.0841 mM and Vmax = 3.6 × 10-7 MS-1), exhibiting a rapid balance period of < 100 s. N-PDA@Mn could activate dissolved oxygen to primarily generate singlet-oxygen (1O2), which oxidized colorless 3,3′, 5, 5′-tetramethylbenzidine (TMB) to blue oxidized (ox-TMB). Because GSH could rapidly deplete 1O2, an ultrafast and colorimetric strategy was developed for GSH detection based on N-PDA@Mn. The GSH detection range was 0.5–100 μM, and the detection limit was 0.28 µM. Importantly, at room temperature, the detection time was only 30 s, which was short than that of previously reported GSH detection methods. Furthermore, a point-of-care testing (POCT) platform was designed by combining the N-PDA@Mn colorimetric sensor with a smartphone. Use the POCT platform, a broad linear detection range of 0.5–100 μM and a detection limit of 0.40 μM were obtained. Subsequently, the N-PDA@Mn colorimetric sensor and POCT platform were successfully used in the real-time and on-site quantification of GSH in human serum, achieving a high accuracy and good recovery. Therefore, the N-PDA@Mn-based methods could realize the rapid, on-site visualization, and economical detection of GSH, demonstrating their substantial potential for application in practical testing in fields of food and medicine.