Abstract We describe here a novel real-time cell tracking system which can measure cell migration routes under cell culture condition. This system includes a mini incubator which controls temperature and CO 2 gas flow and a PDMS (polydimethylsiloxane) chip for chemotaxis measurement. The main differences from previous ones are real-timely long-term (⩾24 h) tracking for single cell quantitatively, simple and inexpensive constitution of optical parts for illumination and imaging, and compatible to commercial well plate. The tracking principle is to trace cell images for each 0.2 s by converting the live cell images to binary images of black and white. Migration results of HUVEC and NCI-H23 cells are obtained respectively using this system. The results are single cell path ( x, y) during migration, cell size, migration distance, migration speed, real-time pictures and so on. This system is applicable to all kinds of researches related to cell migration such as cell angiogenesis, chemotaxis, and moreover cancer metastasis.