Hypoxia plays an important role in tumor progression, and the development of efficient methods for monitoring hypoxic degree in living systems is of great biomedical importance. In the solid tumors, the nitroreductase level is directly corresponded with the hypoxic status. Many one-photon excited fluorescent probes have been developed for hypoxia imaging in tumor cells via the detection of nitroreductase level. However, two-photon excited probes are more suitable for bioimaging. In this work, a two-photon probe 1 for nitroreductase detection and hypoxic status monitoring in living tumor cells and tissues was reported for the first time. The detection is based on the fact that the nitro-group of probe 1 could be selectively reduced to an amino-group by nitroreductase in the presence of reduced NADH, following by a 1,6-rearrangement-elimination to release the fluorophore, resulting in the enhancement of fluorescence. The probe exhibited both one-photon and two-photon excited remarkable fluorescence enhancement (∼70-fold) for nitroreductase, which afforded a high sensitivity for nitroreductase, with a detection limit of 20 ng/mL observed. Moreover, the applications of the probe for fluorescent bioimaging of hypoxia in living cells and two-photon bioimaging in tissues were carried out, with tissue-imaging depths of 70-160 μm observed, which demonstrates its practical application in complex biosystems.