The anticancer therapy strategy mediated by tumor-targeting bacteria needs better visualization tools for imaging and monitoring bacteria in vivo. The probiotic strain Escherichia coli Nissle 1917 (EcN), one of the tumor-targeting bacteria, leads to the potential application for cancer therapy. Here, we report the development and application of a live, EcN-based imageable vehicle for noninvasive in vivo bioluminescence imaging in live mice. Firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE), an enzyme that contributes to stable bioluminescence, were functionally coexpressed in EcN. The recombinant EcN strain expressing the genomically integrated Fluc-LRE cassette was demonstrated to be a valuable tool for generating robust, continuous, and red-shifted bioluminescence for bacterial tracking in vitro and in vivo, thus providing an optical tumor-targeting system for the in vivo study of bacteria-assisted cancer therapy. Additionally, in vivo imaging of the recombinant EcN strain in the mouse intestinal tract indicated the potential of this strain to be used as a tool in the study of gut.