Peroxynitrite (ONOO− ) contributes to oxidative stress and neurodegeneration in Parkinson’s disease (PD). Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO ⁻ flux and understanding of the ONOO ⁻ stress-induced neuropathology of PD. Herein, a novel α-ketoamide-based fluorogenic probe (DFlu) was designed for ONOO ⁻ monitoring in multiple PD models. The results demonstrated thatDFlu exhibits a fluorescence turn-on response to ONOO ⁻ with high specificity and sensitivity. The efficacy ofDFlu for intracellular ONOO ⁻ imaging was demonstrated systematically. The results showed thatDFlu can successfully visualize endogenous and exogenous ONOO ⁻ in cells derived from chemical and biochemical routes. More importantly, the two-photon excitation ability ofDFlu has been well demonstrated by monitoring exogenous/endogenous ONOO ⁻ production and scavenging in live zebrafish PD models. This work provides a reliable and promising α-ketoamide-based optical tool for identifying variations of ONOO ⁻ in PD models.