Microfluidic chips offer the unique opportunity to establish novelty in vitro cells models where the in vivo cells microenvironment could be precisely reconstituted. Although they have significances in the fields of cell biology, the real applications are extremely limited by ambient materials for cells culture, i.e., cells morphology and migration are greatly influenced by substrate material. In this study, we investigated the cells migration effects in four kinds of microfluidic chips with the same geometry. They are PDMS mold structures bonded on culture dish, glass slide, and PDMS substrates, respectively, and another PMMA mold structure bonded on PMMA substrate. For convenient description, we denoted these four chips as PDMS-DISH, PDMS-GLASS, PDMS-PDMS, and PMMA-PMMA, respectively. We compared and summarized the relationship of cells migration effects on different substrate. The cells are initially introduced into the culture area. The experiment results indicate that cells spreading time, spreading area and cells migration on these chips has obvious diversities. To further investigate the cells migration in these chips, a new model is prepared using trypsin/EDTA solution and cell culture medium. It shows a good repeatability. Most of cells could be formed a good morphology and monolayer growth in these microfluidic chips. The cells migrated furthest is in the PDMS-DISH chip after monitored 24 hours. The migration rates are 20.30 μm/h, 18.63 μm/h, 15.00 μm/h, and 10.75 μm/h in PDMS-DISH, PDMS-GLASS, PDMS-PDMS, and PMMA-PMMA, respectively. This study turns open up opportunities for new biochips in prospective applications of wound healing and antiscarring expected in drug screening and the related fields.