We report a mitochondria-targeted near-infrared probe (NRh-O) for frequency upconversion luminescence (FUCL) imaging of hypoxia. Under hypoxic conditions, NRh-O rapidly responds to release the FUCL product NRh (λex/em = 850/825 nm) with high sensitivity and selectivity in mitochondria. This highlights the potential application of a hypoxia-responsive probe in early clinical diagnosis.