While a number of demonstrations have shown bacterial cancer targeting in animal models, the actual mechanism for cancer targeting remains unclear. To detect bacterial preference for cancer cells to normal cells and to elucidate cancer targeting mechanism, a simple co-culture micro-platform has been developed for in vitro studies. Since two different cells types require independent culture environment prior to introducing bacteria, we designed of a passive stop valve at the exit of each cell chamber, providing independent environment for each cell line. When fluorescence tagged bacteria in PBS is introduced into the core of the channel bounded by layers of diluted collagen solutions, the meniscus held at the end of the stop valve breaks. When the flow is stopped, bacteria, initially kept at the core layer, will diffuse into other layers enabling bacteria to move towards cells. We demonstrated a feasibility of this system for bacterial cancer targeting using breast and liver cell lines.