Ferric ion (Fe3+) plays a vital role in cellular homeostasis. However, the detection of Fe3+ with rhodamine B (RhB) has potential problems, such as poor selectivity and low photostability. To address these problems, we rationally designed an RhB@MOF nanocomposite-based "on-off-on" fluorescent switching nanoprobe for highly sensitive and selective detection of Fe3+ and ascorbic acid. This RhB@MOF nanoprobe was prepared through a facile one-pot synthesis. Here MOF served as a selectivity regulator for the detection of Fe3+. By embedding RhB into the porous crystalline MOF, enhanced photostability and fluorescence lifetime of RhB to Fe3+ were achieved. The as-prepared RhB@MOF was demonstrated to be an ultrasensitive and selective nanoprobe for the detection of Fe3+ in human serum and ascorbic acid in rat brain microdialysate. Furthermore, inner filter effect (IFE) and photoinduced electron transfer (PET) were proposed and discussed to explain the selectivity and sensitivity of RhB to Fe3+ against other interfering substances. Our novel "on-off-on" nanoprobe provides insight into the rational design of MOF-based biosensors for selective and sensitive detection of analytes.