A low-temperature carbonization method using seaweed (Sargassum carpophyllum) as a precursor was applied to prepare nitrogen and sulfur co-doped CDs (N,S-CDs). The N,S-CDs exhibited satisfactory stability, water solubility and photostability. The N,S-CDs-based “on–off” fluorescent sensor showed high selectivity and good response to Cr(VI), whose limit of detection (LOD) was as low as 1.04 μM. The recoveries of Cr(VI) in tap water and sea water were 88.90–116.38% (RSD < 5%), proving that it could be a practical sensor for detecting Cr(VI). In addition, the established N,S-CDs/Cr(VI) system was developed as an “off–on” fluorescent sensor to detect ascorbic acid (AA). It could precisely detect AA in vitamin C chewable tablets, with recoveries from 96.02 to 103.95% (RSD < 5%). Moreover, the N,S-CDs expressed low toxicity by CCK8 assay. In this work, an “on–off–on” fluorescent sensor was prepared from seaweed, which can act as a reference for enabled high-value transformation of seaweeds. Furthermore, it also provided a facile fluorescent sensor for the detection of Cr(VI) and AA. The fluorescent sensor expressed high potential in water quality monitoring and nutritional food quality control.