Carbon dots (CDs)-based ratiometric fluorescence strategies have attracted considerable attention. In this study, we developed a facile ratiometric fluorescent biosensor for DNA detection by utilizing the blue-emitting carbon dots (BCDs) as the fluorophore and acriflavine (AF), a fluorescent biological stain reagent, as both the response and recognition molecules. When AF was added, the fluorescence of BCDs decreased effectively and accompanied by a hypsochromic shift from 448 to 425 nm, whereas the fluorescence of AF at about 500 nm appeared due to the synergistic effect of Förster resonance energy transfer (FRET) and the inner filter effect (IFE) between the BCDs and AF. In the presence of DNA, the intercalative binding of AF into the DNA helix resulted in the fluorescence quenching of AF and the fluorescence restoration of BCDs at the same time. Therefore, the quantitative detection of DNA can be carried out vis evaluating the fluorescence intensity ratio of F/F. Under optimized conditions, the developed fluorescence method had an excellent linear range from 0 to 90 µM and a low detection limit of 0.52 µM. In addition, satisfactory results were obtained for the analysis of DNA in synthetic and real samples, with spiked recoveries ranging from 90.1 to 101.3%, demonstrating the enormous potential of the developed sensing platform for bioanalysis and biodetection.