Breast cancer remains a leading cause of mortality among women, driving the need for more accurate diagnostic tools. To address this, a smart ruthenium (Ru)-catalyzed bioorthogonal activation chemiluminescent (BAC) probe has been developed for long-lasting non-invasive in vivo imaging. Although chemiluminescence imaging offers ultrahigh sensitivity without background autofluorescence, its application in breast cancer is limited by poor selectivity in complex tumor environments, slow activation kinetics and insufficient resolution. The BAC probe overcomes these challenges via a smart “Ru-locked” mechanism, achieving light-independent, thiol-triggered activation in complex tumor microenvironment. This mechanism enables rapid activation (1 min), prolonged half-life ( t _1/2 = 18.5 h), and high sensitivity (LOD = 87 nM) across a broad emission range (400-800 nm), while enhancing selectivity for thiol-containing metabolites, particularly H ₂S. The probe exhibits low toxicity in vitro and efficiently activates chemiluminescence within the tumor microenvironment in vivo, enabling precise imaging for tumor localization and image-guided surgery. Additionally, the phenoxy-adamantyl-1,2-dioxetane luminophores are developed via an efficient synthetic route, which reduces the synthesis from seven steps to two, lowering production costs (300- to 400-fold) and increasing yields from 40% to 95%. This study introduces a smart Ru-locked BAC probe for real-time, non-invasive monitoring of thiol-related homeostasis in breast cancer, with promising application in clinical diagnostic and therapeutic potential.