Cancer cell motility and its heterogeneity play an important role in metastasis, which is responsible for death of 90% of cancer patients. Here, in combination with a microfluidic technique, single-cell tracking, and systematic motility analysis,we present a rapid and quantitative approach to judge the motility heterogeneity of breast cancer cells MDA-MB-231 and MCF-7 in a well-defined three-dimensional (3D) microenvironment with controllable conditions. Following this approach identification of highly mobile active cells in a medium with epithelial growth factor will provide a practical tool for cell invasion and metastasis investigation of multiple cancer cell types, including primary cells. Further, this approach could potentially become a speedy (∼hours) and efficient tool for basic and clinical diagnosis.