The pathogenesis of acute kidney injury (AKI) is a multifaceted process involving various mechanisms, with oxidative stress playing a crucial role in its development. Hypochlorite (HOCl) and cysteine (Cys) are indicators of oxidative stress in AKI pathophysiology, directly reflecting the degree of oxidative stress and disease severity. However, their exact mechanism of action in AKI pathophysiology remains unknown. Herein, we developed a dual-channel fluorescent probe, MB-NAP, which allowed for the simultaneous detection of HOCl and Cys. The probe exhibited distinct fluorescence responses in the green channel (λex = 450 nm, λem = 560 nm) and red channel (λex = 610 nm, λem = 690 nm), without any spectral crosstalk, allowing for accurate measurement of both analytes. We successfully applied MB-NAP to monitor the levels of HOCl and Cys in cellular and in vivo models of AKI, revealing a significant increase in their concentrations compared to normal models. Furthermore, MB-NAP was demonstrated to exhibit outstanding capabilities for drug screening by effectively real-time monitoring HOCl and Cys. This study not only provides a more sensitive and reliable method/tool for tracking AKI-related pathological processes but also offers a potential breakthrough in the early diagnosis and identification of therapeutic agents aimed at mitigating oxidative stress-induced damage in AKI.