Peroxynitrite (ONOO⁻) is a potent bio-oxidant involved in many physiological and pathological processes; however, most of the pathological effects associated with ONOO⁻in vivo are still ambiguous. Herein, we designed and synthesized two near-infrared ratiometric fluorescent probes, Ratio-A and Ratio-B, for the detection and biological evaluation of ONOO⁻. The recognition unit diene in the probes could be specifically cleaved by ONOO⁻ with a 94-fold enhancement in the ratiometric fluorescence signal. By imaging ONOO⁻ in immune stimulated cells and acute inflammation mice model using Ratio-A, we investigated the fluctuations of ONOO⁻ levels in a rheumatoid arthritis (RA) model of mice. Ratio-A could be applied for the effective imaging of RA and could rapidly evaluate the response of the RA treatment with methotrexate (MTX). Thus, Ratio-A can be considered as a promising tool for pathological diagnosis and the therapeutic assessment of a wide range of diseases including RA.