Red-to-near-infrared (NIR) fluorophores are highly desirable in bio-imaging studies with advantages of high tissue penetration ability and less interference from auto-fluorescence. However, their preparation usually requires tedious synthetic procedures, which seriously restrict their applications. Thus, the direct preparation of red-to-NIR fluorophores from easily available substrates is highly desirable. Compared with the conventional closed-shell fluorophores, radical cations feature a large red-shift absorption, but only very few of them are fluorescent and they suffer from high instability. Herein, we proposed a convenient strategy for the preparation of red-to-NIR fluorophores through air oxidation of electron-rich 2,5-dimethylpyrroles to in situ generate red-to-NIR emissive radical cations, which can be stabilized by adsorption on silica gel-coated thin layer chromatography (TLC) plates or encapsulated in cucurbit[7]uril (CB[7]). The radical cations derived from pyrroles were verified using electron paramagnetic resonance (EPR) spectroscopy, theoretical calculations and one-electron oxidation experiments. Moreover, the pyrrole-derived radical cations encapsulated in CB[7] can be used for mitochondrial imaging in living cells with high specificity and in vivo imaging with long-term stability. The easily available pyrrole-derived radical cations with red-to-NIR emission are thus promising for biomedical applications.