Doxorubicin (DOX) is a potent anticancer drug, but it has side effects on normal tissues, particularly myocardial cells. Therefore, it is crucial to detect the DOX concentration in body fluids for effective clinical treatment. In this work, N,Bi-codoped CDs (Bi,N-CDs) were synthesized through a one-step hydrothermal method to carbonize the raw materials of 2,4-dinitroaniline and bismuth nitrate. The resulting Bi,N-CDs showed a reduced emission at 490 nm and an enhanced emission at 590 nm in the presence of DOX. The ratio of fluorescence (FL) intensity (F 590 /F 490 ) was found to be a reliable indicator of DOX concentration, ranging from 0.05 to 30 μM and 40−200 μM, with detection limits (LOD) of 34 and 24 nM, respectively. A ratiometric fluorescence nanoprobe was established for highly selective and sensitive detection of DOX using a specific electrostatic interaction and inner filter effect between Bi,N-CDs and DOX. Meanwhile, Bi,N-CDs exhibited a distinct color change ranging from yellow to orange-red when exposed to DOX, allowing for a colorimetric method to measure DOX levels in the range of 0.05−30 μM, with a detection limit of 169 nM. The probe was triumphantly used to monitor DOX in actual samples via a dual-mode optical sensing strategy. This study contributes to the development of heteroatom-doped CDs and expands their potential applications for detecting biological samples.