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Nitric oxide (NO) plays important roles in both physiology and therapeutics. Nonetheless, the in-situ monitoring of NO release from most existing donors is challenging, inhibiting advanced research in both chemistry and biology. In this study, we introduce an innovative photochemotherapeutic agent that incorporates a nitroaniline NO moiety with an integrated pyrene fluorophore. This design enables controlled NO release upon light irradiation, allowing for precise quantification through a dramatic fluorescence enhancement (with a 3000-fold increase in intensity). Ultrafast spectroscopy data revealed that the NO release process is exceptionally rapid, completing within 13.3 ps. Additionally, the donor's fluorescence was successfully utilized for in situ cell imaging upon direct photoexcitation. Consequently, using this fluorescent probe enables the accurate visualization of NO release kinetics, dosage, and localization in real-time. The unique advantages of this NO donor are anticipated to significantly advance research in NO chemistry and biology. [Display omitted] • The first pyrene-based photoactivated NO donor was fabricated. • Photoactive ultrafast NO release (13.3 ps) was obtained. • Real-time NO-releasing was monitored by a strong fluorescent probe (3000-fold). • Cellular imaging was performed to demonstrate NO-releasing in situ. [ABSTRACT FROM AUTHOR]