Hydrogen peroxide is one of the most important reactive oxygen species, which plays a vital role in many physiological and pathological processes. A dramatic increase in HO levels is a prominent feature of cancer. Therefore, rapid and sensitive detection of HO in vivo is quite conducive to an early cancer diagnosis. On the other hand, the therapeutic potential of estrogen receptor beta (ERβ) has been implicated in many diseases including prostate cancer, and this target has attracted intensive attention recently. In this work, we report the development of the first HO-triggered ERβ-targeted near-infrared fluorescence (NIR) probe and its application in imaging of prostate cancer both in vitro and in vivo. The probe showed good ERβ selective binding affinity, excellent HO responsiveness and near infrared imaging potential. Moreover, in vivo and ex vivo imaging studies indicated that the probe could selectively bind to DU-145 prostate cancer cells and rapidly visualizes HO in DU-145 xenograft tumors. Mechanistic studies such as high-resolution mass spectrometry (HRMS) and density functional theory (DFT) calculations indicated that the borate ester group is vital for the HO response turn-on fluorescence of the probe. Therefore, this probe might be a promising imaging tool for monitoring the HO levels and early diagnosis studies in prostate cancer research.