Adoptive T cell therapy that collected from the patient’s blood for treating cancer and chronic infections has revolutionized the field of oncology and personalized medicine. However, such highly activated T cells that have specific T cell receptor (TCR) against the target disease are usually greatly outnumbered by not-activated T cells due to the stochastic generation of TCR by V(D)J recombination, approximately 10⁶∼10⁷ possible TCR. In this report, for the purpose of representing the lymph node where T cells can migrate on the surface of the antigen presenting cells (APCs) for activation or sequentially interact to multiple APCs, an open-type PDMS microfluidic device has been developed. This device mimics the microenvironment of a lymph node for the T cells to scan, contact and interact with APC and has the structure for the plan to collect the cells and conduct downstream analysis later on. By measuring and profiling Ca2+ flux of the T cell that interacted with APC, the threshold classifying whether T cells activation is specific or not was calculated. This new tool is expected to be useful in providing new insight and strategy to basic biology.