It is of great significance to develop an eco-friendly and sensitive platform for the detection of 6-mercaptopurine (6-MP) because of its side effects and variable activity. Herein, fluorescence quenching and spectrophotometric methods for the detection of 6-mercaptopurine (6-MP) using carbon dots (CDs) as a fluorescence probe are established. 6-MP not only serves to shelter the CDs effectively from being quenched, but also to reverse the quenching and restore the fluorescence due to its ability to remove Hg²⁺ from the surface of CDs. After the experimental conditions are optimized, the linear range for the detection of 6-MP is 0.04 to 12 μmol L⁻¹, and the detection limit is 0.01 μmol L⁻¹. When it refers to the spectrophotometric method, the linear range is 0.08 to 12 μmol L⁻¹ and the detection limit is 0.02 μmol L⁻¹. The CDs with 6-MP are systematically characterized with transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) absorption and so on. Furthermore, the proposed methods are not only expected to become a potential tool for the fast response of 6-MP but also possess the potential for practical application to be applied in the determination of 6-MP in human serum samples with satisfactory results.