Recently, sensitive and selective detection of exosomal microRNAs (miRNAs) has been garnering significant attention, because it is related to many complex diseases, including cancer. Herein, we report a ratiometric fluorescent bioprobe based on DNA-labeled carbon dots (DNA-CDs) and 5,7-dinitro-2-sulfo-acridone (DSA) coupling with the target-catalyzing signal amplification for the detection of exosomal miRNA-21. There was high fluorescence resonance energy transfer (FRET) efficiency between carbon dots (CDs) and DSA when the bioprobe was assembled. However, in the presence of the target, with disassembling of the fluorescent bioprobe, the fluorescence intensities of CDs and DSA were changed simultaneously. Because of the ratio of dual fluorescence intensities, this ratiometric fluorescent bioprobe was able to cancel out environmental fluctuations by calculating emission intensity ratio at two different wavelengths, being robust and stable enough for detection of exosomal miRNA-21. In addition, we displayed that a single miRNA-21 can catalyze the disassembly of multiple CDs with DSA theoretically, yielding significant change in the fluorescence ratio for the detection of miRNA-21. With this signal amplification strategy, the limit of detection was as low as 3.0 fM. Furthermore, because of the introduction of lock nucleic acid to mediate the strand displacement reaction, the selectivity of this strategy was improved remarkably, even against single base mismatch sequence. More importantly, our strategy could monitor the dynamic change of exosomal miRNA-21, which maybe becomes a potential tool to distinguish cancer exosomes and nontumorigenic exosomes. In a short, this ratiometric fluorescence bioprobe possessed high stability, sensitivity and selectivity coupling with ease of operation and cost efficiency, leading to great potential for wide application.