Metastasis is an important hallmark of malignant tumors. In this study, we developed a microfluidic system to screen highly metastatic sublines via differential resolution of cell invasiveness. The system was composed of a PDMS-glass device connected with a syringe pump and a Petri dish. To facilitate the selection process, a long-term cell invasion driving force based on a chemotactic factor gradient was created using the Petri dish-based liquid supply pattern, and the invasive cells were collected for round-by-round selection via an open region in the chip. Using the system, we established an SGC-7901/B2 subline from the human gastric cancer SGC-7901 cell line by only two rounds of selection. In vitro assays showed that the SGC-7901/B2 cells were superior to the parental cells in proliferation and invasiveness. Furthermore, an in vivo tumorigenicity assay demonstrated that compared with the parental cells, the subline had stronger spontaneous metastatic and proliferative capability, which led to a shorter survival duration. Additionally, the protein expression differences including E-cadherin and Smad3 between the subline and parental cells were revealed. In conclusion, this microfluidic system is a highly effective tool for selecting highly metastatic sublines, and SGC-7901/B2 cells could serve as a potential model for tumor metastasis research.