Developing probes with good biocompatibility and realizing intracellular detection in living cells are of great significance for biomedicine and life sciences, but remain a challenge presently. In this paper, we describe a rapid and highly selective biosensor for Fe3+ detection in living cells based on the Maillard reaction fluorescent products (MRFPs) of glutathione and ascorbic acid as a probe. Experiments show that the MRFPs are non-cytotoxic and possess excellent biocompatibility. Moreover, the MRFPs show a rapid response and good selectivity towards Fe3+ over other metal ions under physiological pH conditions in vitro. The introduction of Fe3+ can quench the fluorescence of MRFPs, and the fluorescence intensity of system decreases linearly with the increasing concentration of Fe3+ in the range of 0.05-50 μM with the detection limit of 4.6 nM at a signal-to-noise ratio of 3. Moreover, the recognition mechanism has been discussed, which is attributed to the charge transfer from excited-state MRFPs molecules to metal ions. In addition, the MRFPs have been successfully demonstrated to be a good imaging probe for Fe3+ sensing in living cells. This study shows that the biocompatible MRFPs might hold great potential for applications in bioimaging, diagnosis, and therapy of intracellular diseases.