Iron ions, as a main component of intracellular labile iron, not only play an important function in oxygen transport, enzymatic reactions, and electron transport but also are vitally important in oxidative stress. In this work, we developed a ratiometric fluorescent biosensor for ferrous ion (Fe2+), in which gold nanoclusters (AuNCs) were synthesized as a stable fluorescent probe and a ligand (FeL) was designed for specific recognition of Fe2+ and conjugated onto AuNCs (AuNC@FeL). Meanwhile, water-soluble sulfocyanine 7 N-hydroxysuccinimide ester (Cy7 NHS ester) was immobilized onto AuNC@FeL as a reference element. The developed ratiometric fluorescent nansosensor displayed good linearity with the concentration of Fe2+ in the range of 1-105 μM, and detection limit was achieved down to 210 nM. In addition, this nanosensor responded to Fe2+ in less than 1.23 s and showed high selectivity against other metal ions, amino acids, and reactive oxygen species. With the advantages of high selectivity and accuracy, as well as quick response and long-term stability, this organic-inorganic ratiometric fluorescent probe was successfully applied in real-time biosensing and bioimaging of Fe2+ in neurons and HepG2 cells. By use of this tool, it was found that the increasing concentration of Fe2+ in live cells was closely related to oxidative stress.