摘要： Chondroitin sulfate (CS) plays an increasingly important role in clinical settings and pharmacy quality control. However, sensitive and simple methods for CS detection remain limited. In this work, positively charged nitrogen doped carbon dots (P-NCDs) with internal luminescence and quenching property to FAM-labeled random-sequence ssDNA (F-ssDNA) were prepared by a simple heating method. P-NCDs attached and quenched F-ssDNA through electrostatic interaction to form the system of P-NCDs and F-ssDNA (P-NCDs/F-ssDNA) with retained fluorescence intensity of P-NCDs. The highly negatively charged CS reacted electrostatically with P-NCDs and then replaced F-ssDNA in P-NCDs/F-ssDNA to recover the fluorescence intensity of the original quenched F-ssDNA while retaining the internal fluorescence intensity of P-NCDs. Thus, by using restored F-ssDNA as the signal controlled by adding CS to P-NCDs/F-ssDNA, a ratiometric fluorescence strategy based on the retained fluorescence of P-NCDs as reference signal was fabricated through synchronous fluorescence spectrometry for the sensitive detection of CS. Under the optimal experimental conditions, a linear equation for CS was obtained for CS concentration within the range of 0.05–2.0 μg/mL. The method was also successfully applied for the accurate determination of CS in joint fluid samples of arthritic patients, chondroitin sulfate tablets, and chondroitin sulfate eye drops, suggesting its appreciable application potential in the clinic.