In this manuscript, a novel probe RHBI based on the rhodamine-benzimidazole conjugate was designed and synthesized. RHBI showed an extreme selectivity for Fe3+ over other metal ions such as Pb2+, Ni2+, Co2+, Mn2+, Zn2+, Hg2+, Cd2+, Ag+, Mg2+, Ca2+, Ba2+, Na+ and K+ in acetonitrile. Upon the addition of 10 equiv. of Fe3+, a 1098-fold fluorescence intensity enhancement was observed at the maximum emission wavelength of 582 nm. Both the Job’s plot and ESI-MS showed that RHBI coordinated with Fe3+ in a 1:1 stoichomitry and the calculated binding constant was 1.01 × 104 M−1. The competition experiment for Fe3+ ions mixed with other metal ions exhibited no obvious change except Cu2+ that could induce a mild fluorescence quenching. Moreover, the fluorescence emission increased linearly with the Fe3+ concentration in the range of 6 × 10−6–4 × 10−5 M and the detection limit was 1.5 × 10−8 M.