Ratiometric fluorescence imaging can decrease or even eliminate the influences of the microenvironment, localized probe distribution, and instrumental parameters, thereby permitting more accurate monitoring of intracellular molecular events. Therefore, developing ratiometric fluorescent nanoprobes is highly important. In this study, we proposed a distinctive approach by combining solvent extraction with a solvothermal method to synthesize novel biomass-derived carbon-based quantum dots (termed as biomass quantum dots, BQDs) with single-excitation and dual-emission properties, using chlorophyll extracted from pakchoi and polyoxyethylene bisamine as raw materials. The obtained BQDs emit blue and red emissions centered at 488 nm and 678 nm respectively under single 413 nm excitation. Low cytotoxicity and unique optical features make the obtained BQDs potentially useful in bio-application as ratiometric fluorescent probes. Taking advantage of these merits, we employ the obtained BQDs for the near-infrared ratiometric fluorescence detection of coenzyme A (CoA) by the assistance of copper ions. This ratiometric approach shows a good sensitivity and selectivity, and the ratiometric fluorescence imaging of CoA in living cells is also achieved.