A highly sensitive Tb(3+) complex-based luminescent probe, N,N,N(1),N(1)-[2,6-(3'-aminomethyl-1'-pyrazolyl)-4-(3'',4''-diaminophenoxy)methylene-pyridine] tetrakis(acetate)-Tb(3+) (BMTA-Tb(3+)), has been designed and synthesized for the recognition and detection of hydrogen peroxide (H(2)O(2)) in aqueous solutions. This probe is almost nonluminescent because the Tb(3+) luminescence is effectively quenched by the electron-rich moiety, diaminophenyl, on the basis of the photoinduced electron transfer (PET) mechanism. In the presence of peroxidase, the probe can react with H(2)O(2) to cause the cleavage of the diaminophenyl ether, which affords a highly luminescent Tb(3+) complex, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-hydroxymethyl-pyridine] tetrakis(acetate)-Tb(3+) (BHTA-Tb(3+)), accompanied by a 39-fold increase in luminescence quantum yield with the increase of luminescence lifetime from 1.95 to 2.76 ms. The dose-dependent luminescence enhancement of the probe shows a good linearity with a detection limit of 3.7 nM for H(2)O(2), which is approximately 14-fold lower than those of the commonly used fluorescent probes. The probe was used for the time-resolved luminescence imaging detection of the oligosaccharide-induced H(2)O(2) generation in tobacco leaf epidermal tissues. On the basis of the probe, a background-free time-resolved luminescence imaging method for detecting H(2)O(2) in complicated biological systems was successfully established.