Notum, one of the key serine hydrolases in mammals, hydrolyzes the palmitoleoyl moieties of many important proteins and modulates multiple signaling pathways including Wnt/β-catenin signaling. Notum is tightly associated with multiple human diseases, but the reliable and practical tools for sensing Notum activities in complex biological systems are rarely reported. Herein, an efficient strategy was used to rationally construct a specific bioluminescent substrate for Notum. Following computer-aided molecular design and experimental verification, octanoyl luciferin (OL) was selected as the optimum substrate for human Notum, with excellent specificity, high detection sensitivity and high signal-to-noise ratio. Under physiological conditions, OL was readily hydrolyzed by Notum or Notum-containing biological specimens to release d-luciferin that could be easily detected by various fluorescence devices in the presence of luciferase. The applicability of OL for real-time sensing native Notum was examined in living cells, extracellular matrix, and tissue preparations. OL was also used for constructing a high-throughput assay for screening of Notum inhibitors, while a natural compound (bergapten) was newly identified as a potent Notum inhibitor. Collectively, this study devises a reliable and easy-to-use tool for sensing Notum activities in biological systems, which will strongly facilitate hNotum-associated fundamental studies, disease diagnosis, and drug discovery.