A fluorescent probe (COU-LIP-1) for monitoring labile Fe(II) pools (LIP) with high selectivity and sensitivity was developed utilizing coumarin 343 as the fluorophore and 3-nitrophenylazanyl ester as both the reactive group and the fluorescence quenching group. Fe(II)-induced reductive cleavage of the N-O bond results in a turn-on response via a photo-induced photon transfer (PET) mechanism. The probe was applied for monitoring labile iron(II) changes in M1 and M2a macrophage activations and also erastin-induced ferroptosis, providing a powerful tool for selectively sensing LIP under both physiological and stressed conditions.