Tirapazamine (TPZ), an antitumor prodrug, can be activated in hypoxic environment. It specifically targets the hypoxic microenvironment of tumors and produces toxic free radicals. However, due to the tumor is not completely hypoxic, TPZ often fails to effectively treat the entire tumor tissue, resulting in suboptimal therapeutic outcomes. Herein, a low pathogenic Escherichia coli TOP10 is utilized to selectively colonize tumor tissues, disrupt blood vessels, and induce thrombus formation, leading to the expansion of hypoxic region and improving the therapeutic effect of TPZ. Additionally, a thermosensitive hydrogel is constructed by Pluronic F‐127 (F127), which undergoes gelation in situ at the tumor site, resulting in sustained release of TPZ. To monitor the therapeutic process, it is genetically modified TOP10 by integrating the bioluminescent system luxCDABE (TOP10‐Lux). The bioluminescent signal is associated with tumor hypoxia enhancement and thrombus formation, which is beneficial for therapeutic monitoring with bioluminescence imaging. In the murine colon cancer model, the TOP10‐Lux combined with TPZ‐loaded F127 hydrogel effectively suppressed tumor growth, and the treatment process is efficiently monitored. Together, this work employs genetically modified TOP10‐Lux to enhance the therapeutic efficacy of TPZ and monitor the treatment process, providing an effective strategy for bacteria‐based tumor‐targeted chemotherapy and treatment monitoring.