Glutathione peroxidase 4 (GPx4) is the membrane peroxidase in mammals that is essential for protecting cells against oxidative damage and critical for ferroptosis. However, no live cell probe is currently available to specifically label GPx4. Herein, we report both inhibitory and noninhibitory fluorescent turn-on probes for specific labeling of GPx4 in live cells. With these probes, the GPx4 expression levels and degradation kinetics in live cells could be visualized, and their real-time responses to the cellular selenium availability were revealed. These probes could also potentially serve as staining reagents to predict the sensitivity of GPx4-related ferroptosis drugs. In view of these features, these GPx4-selective probes will offer opportunities for a deeper understanding of GPx4 function in natural habitats and hold great promise for biomedical applications.