Two novel theranostic agents <b>HJTA</b> and <b>HJTB</b> have been designed and synthesized by covalently linking a β-carboline derivative, with antitumor activities and pH-responsive fluorescence, with a 2-exomethylenecyclohexanone moiety, which can be activated by the tumor-targeting glutathione (GSH)/glutathione S-transferase π (GSTπ). These agents showed pH- and GSH-dual-responsive fluorescence in tumor cells but not in normal cells. Importantly, <b>HJTA</b> selectively illuminated tumor tissue for up to 7 h and generated precise visualization of orthotopic colonic tumors through the blood circulation system in intraoperative mice. Furthermore, <b>HJTA</b> exhibited potent and selective antiproliferative activities and colonic tumor inhibition in mice. Finally, <b>HJTA</b> induced great cancer cell apoptosis and autophagy by regulating the expression of apoptotic and autophagic proteins. Therefore, this pH/GSH-dual-responsive fluorescent probe with cancer-targeting therapeutic activity provides a novel tool for precise diagnosis and tumor treatment, therefore broadening the impact of multifunctional agents as theranostic precision medicines.