Doping with transition-metal ions is an intriguing way to tune the properties of quantum dots. In this study, water-soluble and stable Ni-doped CdTe quantum dots have been successfully synthesized via a facile, novel and green method. Compared with undoped CdTe quantum dots, the Ni-doped CdTe quantum dots exhibited improved photostability, and lower cytotoxicity and enhanced biocompatibility. A simple, affordable, selective, and sensitive method based on the fluorescence quenching of Ni-doped CdTe quantum dots for rapid determination of pyrazinamide (PZA) in plasma samples was developed. Fluorescence of Ni-doped CdTe quantum dots can be effectively quenched by PZA drug due to the binding of PZA drug on the surface of Ni-doped CdTe quantum. Under the optimized conditions, the response was directly proportional to the concentration of PZA in the range of 2 to 100 μM, with a detection limit of 0. 5 μM. The proposed method was applied to the determination of PZA in plasma samples with satisfactory results.