Two novel theranostic agents HJTA and HJTB 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, HJTA 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, HJTA exhibited potent and selective antiproliferative activities and colonic tumor inhibition in mice. Finally, HJTA 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.