A growing interest has emerged in carbon dot (CDs) fluorescent probes, as they are non-destructive, non-contact, sensitive, fast, and low-cost. However, the low fluorescence quantum yield (QY) and the uncertainty of fluorescence quenching mechanism are the main bottlenecks restricting its application. Here, we used mustard seeds as carbon source, CDs were synthesized by a simple hydrothermal method without further surface treatment with the QY reached a value as high as 17.92 % in 468 nm excitation energy. As a label-free fluorescent probe, CDs can be used to sense Fe3+ ions in environmental water and human oral keratinocytes (Hok) cells, with a detection limit of 0.51 μM and the linear range of 0–75 μM. Moreover, we have conducted an in-depth study on the mechanism of fluorescence quenching of CDs caused by Fe3+ ions. Our study provides powerful evidences that the mechanism of fluorescence quenching to Fe3+ ions is a synergistic effect of static quenching (SQ) and aggregation-induced effect (AIE), which accompanied by forming a non-fluorescent ground state complex between CDs and Fe3+ ions. This work promises to provide new ideas for the most appropriate detection strategy and design the best performing sensor, allowing their effective application in the sensing field.