Bacterial cancer targeting may become an efficacious cancer therapy, but the mechanisms underlying bacterial specificity for cancer cells need to be explored prior to adopting it as a new clinical application. To characterize the mechanism of bacterial chemotactic preference towards cancer cells, we developed a microfluidic device for in vitro study. The device consists of a cell culture chamber on both sides of a central bacteria channel, with micro-channels used as barriers between them. The device, when used as model for lung cancer, was able to provide simultaneous three-dimensional co-culture of multiple cell lines in separate culture chambers, and when used as model for bacterial chemotaxis, established constant concentration gradients of biochemical compounds in a central channel by diffusion through micro-channels. Fluorescence intensity of green fluorescence protein (GFP)-encoding bacteria was used to measure bacterial taxis behavior due to established chemotactic gradients. Using this platform, we found that Escherichia coli (E. coli) clearly illustrated the preference for lung cancer cells (NCI-H460) which was attributed to biochemical factors secreted by carcinoma cells. Furthermore, by secretome analysis and validation experiments, clusterin (CLU) was found as a key regulator for the chemotaxis of E. coli in targeting lung cancer.