Objective: Based on the high affinity between Cu2+ and sulfur atom in clinical commonly used anticancer drug 6-thioguanine (6-TG), a novel ratiometric fluorescence method was constructed for the detection of 6-TG. Methods: o-Phenylenediamine (OPD) could be oxidized by Cu2+ to its yellow fluorescence product 2,3-diaminophenazine (DAP) at 560 nm. The fluorescence intensity of blue-Carbon Dots (b-CDs) at 450 nm could be quenched by DAP because of the inner filter effect (IFE). However, in the presence of 6-TG, it will compete with OPD for binding Cu2+, resulting in the reduction of the oxidation product DAP and its yellow fluorescence. Therefore, the IFE between the b-CDs and DAP was weakened, and the blue fluorescence of the b-CDs was restored. The fluorescence intensity ratio (F450/F560) is linearly correlated with the concentration of 6-TG. Therefore, this probe could be used to quantitatively detect 6-TG. Results: With the increase of 6-TG, the fluorescence intensity ratio (F450/F560) was gradually enhanced. Under the optimal reaction conditions, the linear concentration of 6-TG ranged from 40.00 to 160.0 μM with detection limits of 61.10 nM, and the correlation coefficient R2 was 0.992. Conclusion: This probe possesses good selectivity, high accuracy and is easy to operate. In addition, it is practical and economical, and can be applied to determine the content of 6-TG tablet.