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A ratiometric optical dual-mode probe was first designed for the detection of doxorubicin (DOX) based on 11-mercaptoundecanoic acid-functionalized carbon dots (MUA-CDs). The carbon dots (CDs) were synthesized via hydrothermal carbonization of m-phenylenediamine and p-aminobenzoic acid, and then it was reacted with 11-mercaptoundecanoic acid by the amide reaction to obtain the MUA-CDs. The as-synthesized MUA-CDs have a high fluorescence quantum yield (59.4%) with bright green emission. Based on a specific electrostatic interaction and the fluorescence resonance energy transfer (FRET) between MUA-CDs and DOX, a ratiometric optical dual-mode probe was constructed for the detection of DOX with good selectivity, high sensitivity, and excellent anti-interference performance. When DOX was mixed the MUA-CDs, the fluorescence of the MUA-CDs at 513 nm decreased, while the fluorescence at 590 nm increased. With this information, a ratiometric fluorescent method was established to determine DOX with a linear range and limit of detection (LOD) of 0.25–19.96 and 0.66 μM and 50.33–80.88 and 0.23 μM, respectively. Meanwhile, the color of the MUA-CDs varied from colorless to dark yellow in the presence of DOX, and therefore, a ratiometric colorimetric method was applied to determine DOX in a range of 2.50–29.80 μM with a LOD of 0.75 μM. Finally, the as-constructed probe was successfully used to determine DOX in serum, urine, and live cells with satisfactory results. This work will contribute to the specific design of functional CDs and broaden the application of functional CDs in analytical detection and biological imaging.

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