Iron, nitrogen-co-doped carbon quantum dots (Fe,N-CDs) were prepared via a simple one-step hydrothermal method. The quantum yield of fluorescence reached about 27.6% and the blue-emissive Fe,N-CDs had a mean size of 3.76 nm. The as-prepared carbon quantum dots showed good solubility, a high quantum yield, good biocompatibility, low cytotoxicity, and high photostability. Interestingly, the as-prepared Fe,N-CDs exhibited good selectivity and sensitivity toward both hematin and ferric ions, and the limit of detection for hematin and ferric ions was calculated to be about 0.024 μM and 0.64 μM, respectively. At the same time, Fe,N-CDs were used for imaging HeLa cells and showed that most Fe,N-CDs were detained in the lysosome. Thus, this fluorescent probe has potential application in the quantitative detection of hematin or Fe3+ in a complex environment and for determining Fe3+ at the cellular level.