Ferric(III) ions (Fe3+) are one of the most abundant metal ions in environmental and biological systems. The determination of Fe3+ has attracted great attention for healthcare concerns. In this work, we have developed a novel fluorescence method for the sensing and intracellular imaging of Fe3+ based on the prepared red-emissive carbon nanodots. The nanoprobes are synthesized via a microwave method using ammonium fluoride and o-phenylenediamine as carbon precursors, which exhibit excellent optical properties and low toxicity. More importantly, the carbon nanodots show high selectivity towards Fe3+ against other interfering ions. The sensitivity is also high with the limit of detection as low as 0.05 μM. Meanwhile, the carbon nanodots have been successfully used for fluorescence imaging of cells and could be quenched by intracellular Fe3+. These results suggest that the red-emissive carbon nanodots have diverse potential utilities in biomedical fields.