Tumor metastasis is an important reason for the poor prognosis and high mortality in cancer patients. As major component of stromal cells in tumor microenvironment, cancer-associated fibroblasts (CAFs) secreted various factors to promote tumor metastasis. Studies have indicated that endogenous direct current electric field (dcEF) around tumor tissue induced directional migration of cancer cells. However, the regulatory effect of CAFs on cancer migration under dcEF stimulation is still unknown. In this study, a two-layers polydimethylsiloxane (PDMS)-based microfluidic chip was fabricated. The introduction of concave structures achieved the non-contacted co-culture of different cell types, and parallel channels in the chip provided stable and homogeneous dcEF. Cells electrotactic response was evaluated under co-culture circumstance. The results showed that CAFs exhibited directional migration towards anode under dcEF stimulation, while A549 cells had a trend of directional migration towards cathode. The co-existence of CAFs and dcEF significantly enhanced the motility and cathodal migration of A549 cells, suggesting synergistic influences of chemotaxis from CAFs and electrotaxis from dcEF stimulation. Moreover, we demonstrated that lung normal fibroblasts acquired CAFs properties after stimulated by dcEF, characterizing by increasing gene expression of α-SMA and IL-6. Overall, Our device and study provide new insight for tumor electrotaxis in complex microenvironment.