Herein, a one-step hydrothermal synthesis method was adopted to fabricate carbon dots (CDs) from amido black 10b in a sodium hydroxide solution. The morphology and composition of the CDs were investigated by XRD, FTIR TEM, XPS, UV–vis, and fluorescence spectroscopy. The obtained CDs (AB-CDs) with an average diameter of 19.4 nm displayed a well-dispersed characteristic in aqueous solutions. The as-prepared CDs showed bright blue fluorescence and good photostability, with a high quantum yield of 24.1%. AB-CDs displayed a selective and noticeable turn-off response to Fe3+. Accordingly, the quantitative detection of Fe3+ was achieved in the range of 5–200 μmol L–1 with a detection limit of 1.84 μmol L–1. The fluorescence response mechanism of Fe3+ to AB-CDs was ascribed to static quenching due to the emergence of the ground-state complex. Moreover, ascorbic acid could restore the fluorescence of AB-CDs quenched by Fe3+ by reducing Fe3+ to Fe2+. The developed nanoprobe was used to detect ascorbic acid with a limit of detection of 7.26 μmol L–1 in the range of 20–300 μmol L–1. Furthermore, the developed sensing system was successfully applied for an Fe3+ assay in a lake water sample and ascorbic acid detection in a human urine sample. The AB-CD-based analytical system showed its latent practical value in the chemical analysis and bioanalytical fields.