Two new pyridine-type rhodamine B chemosensors (RBPO and RBPF) used to detect Fe3+ have been designed and synthesized, and the sensing behavior towards various metal ions was evaluated via UV-vis and fluorescence spectroscopic techniques. Both RBPO and RBPF not only have good spectral responses to Fe3+ in an EtOH/H2O solution (3 : 1, v/v, HEPES, 0.5 mM, pH = 7.33) with low detection limits and high binding constants, but also suffer from less interference from common metal cations. The two chemosensors are further proven to be practical in sensitively monitoring trace Fe3+ in real water specimens. Intracellular imaging applications demonstrated that RBPO and RBPF can be used as two fluorescent chemosensors for the detection of Fe3+ in living human breast adenocarcinoma (MCF-7) cells.