Hydrogen peroxide (H₂O₂) is a type of reactive oxygen species that regulates essential biological processes. Despite the central role of H₂O₂ in pathophysiological states, available molecular probes for assessing H₂O₂in vivo are still limited. This work develops hyperpolarized ¹⁵N-boronobenzyl-4-cyanopyridinium (¹⁵N-BBCP) as a rationally designed molecular probe for detecting H₂O₂. The ¹⁵N-BBCP demonstrated favorable physicochemical and biochemical properties for H₂O₂ detection and dynamic nuclear polarization, allowing noninvasive detection of H₂O₂. In particular, ¹⁵N-BBCP and the products possessed long spin–lattice relaxation times and spectrally resolvable ¹⁵N chemical shift differences. The performance of hyperpolarized ¹⁵N-BBCP was demonstrated both in vitro and in vivo with time-resolved ¹⁵N-MRS. This study highlights a promising approach to designing a reaction-based ¹⁵N-labeled molecular imaging agent for detecting oxidative stress in vivo.