The development of NIR fluorescent probes for biomedical applications has outstanding potential because of the lower background fluorescence present involving less scattering and deeper tissue penetration, resulting in limited damage to living cells. Recently, dicyanomethylene-4H-pyran systems have been utilized as an NIR fluorophore for the detection of various analytes and in cell imaging studies. These efforts have involved single-as well as two-photon microscopy. As part of an effort to generate novel probes that can allow biologists/neurobiologists better visualization/quantification of the role of reactive oxygen/nitrogen species in the living system, a new dicyanomethylene-4H-pyran-based fluorescent probe DCPO-DP has been designed, synthesized and characterized. The fluorescence of the probe is pre-quenched by hydroxyl protection (diphenyl phosphinate protecting group). A selective "TURN-ON'' fluorescence response involving an similar to 120-fold emission intensity enhancement was observed upon reaction with ONOO-, compared with emission signals arising from other ROS/RNS. The reaction was completed within 20 min. The detection limit was determined to be 4.62 mu M. Fixed cells were used in imaging experiments and helped demonstrate this novel chemosensing strategy for the detection of ONOO- in living systems with phosphinates.