Ulcerative colitis (UC) is an inflammatory disease that leads to the overexpression of peroxynitrite (ONOO-). Up to now, it has been a challenge to design a colon-targeted fluorescent probe with a large emission shift that can detect and image ONOO- in UC mice. Here, a fluorophore (pyran-coumarin) is linked with cholic acid to develop a colon-targeted fluorescent probe (CPC) for the detection of ONOO-. The fluorescent probe showed strong near-infrared emission at 725 nm. After reacting with ONOO-, it can be observed that the fluorescence intensity at 725 nm decreases obviously, and the signal at 490 nm increases clearly with an obvious emission shift (235 nm). This response mechanism causes the probe to have good sensitivity and selectivity. Additionally, the probe CPC shows good targeting ability for the cells with overexpressingTGR5 receptors and displays good results for detecting exogenous and endogenous ONOO- in the cells. More importantly, the probe CPC can be especially assembled in the colon site to distinguish normal and UC mice and provide significant information for the diagnosis of UC.