Objective To investigate the Force-chemical coupling regulation mechanism of chemokine-induced calcium response in Jurkat T cells under fluid shear stress. Methods With the combination of flow chamber and flourescence microscope calcium response in Jurkat T cells, stained with Fluo-4 AM, was observed with or without extracellular calcium ions under fluid shear stress. Corresponding characteristic parametres were extracted. Results CXCL12 could induce firm adhesion of Jurkat T cells in a concentration-dependent manner. Force was the key to start calcium response. When the fluid shear force increased from 0 to 20 mPa, activation ratio rouse up to 75% from 4%. Under fluid shear stress of 20 mPa, extracellular calcium ions could speed the process of calcium response by shortening delay time (about 23 s), prolong half time (about 7 s) and half time (about 20 s) and strengthen intensity. Conclusions Fluid shear stress and extracellular calcium ions synergize to accelerate and enhance CXCL12-mediated calcium response in Jurkat T cells, suggesting a fast ‘extracellular calcium influx-intracellular calcium store release’ pathway which shows MS dependence. This study can deepen the understanding T cells activation and provide reference for relevant pathological and drug research.