Ferroptosis, a new form of regulated cell death, results from the iron-dependent accumulation of lipid peroxides that are associated with reactive oxygen species. However, it remains unclear how hydroxyl radical ((OH)-O-center dot) and cellular rnicroenvironments such as viscosity alter in this process. Herein, we characterize for the first time the changing behavior of (OH)-O-center dot and cytoplasmic viscosity during ferroptosis using a dual-functional fluorescence probe (H-V) that is designed via the molecular rotor strategy and the unique aromatic hydroxylation of (OH)-O-center dot. Probe H-V shows completely separate spectral responses to (OH)-O-center dot and viscosity with high sensitivity and selectivity, thereby achieving the detection of (OH)-O-center dot and viscosity in two independent channels without spectral cross interference. With the probe we find that ferroptosis is accompanied by significant (OH)-O-center dot generation and cytoplasmic viscosity increase. Most notably, the raised (OH)-O-center dot comprises the majority of the total reactive oxygen species in ferroptosis. H-V is biocompatible, ready to prepare, and may be expected to be used in the study of viscosity and (OH)-O-center dot detection in more biosystems.