<p>Biomolecular detection based on a single fluorescence signal response is often difficult to completely eliminate the influence from interfering factors on the assay results. In this work, a ratiometric fluorescence probe was designed based on a dual emission carbon dot (CD). It produces dual-color emission (400 nm blue emission band and 560 nm green emission band) under 360 nm excitation. The two emission bands are believed to be originated from the distinct luminescent mechanisms: the intrinsic emission of CDs and intramolecular charge transfer (ICT) process, respectively. The energy transfer between the dual emission leads to the differentiated responses towards sialic acid (SA). As the concentration of SA increases, the two emission bands of CDs dispersion exhibit opposite variation trends. The blue emission intensity rose slightly and green emission intensity descended. The ratios of emission band intensity at 400 nm and 560 nm are served as quantitative assay of SA. The limit of detection reached 1.15 μM. The ratiometric fluorescence probe also shows a favorable selectivity for SA detection. The self-calibration and mutual verification functions of the probes enable consistent emission ratios across different models of commercial spectrometers. It minimizes the interference from the environment and instrumentation, achieving accurate and highly versatile SA detection. The utility of the probe for SA detection was verified by analyzing human serum, showing the tremendous potential of dual emission CDs for accurate SA detection.</p> Graphical Abstract <p></p>