Objective Study of the kinetics of extracellular regulated protein kinase (ERK) activity in Jurkat T cells and the effect of matrix stiffness on ERK activity. Methods ERK activity in cells was visualized by fluorescence resonance energy transfer (FRET) biosensor, and cells were embedded into type I collagen (COL) hydrogel to check the biomechanical effect. Results Experiments showed pulsation of ERK activity in a sub-group of Jurkat cells, which had frequency of three times in one hour and averaged ~20% change in oscillating magnitude. T cell receptor (TCR) activation with antibodies didn’t change ERK pulsation in frequency or magnitude. ERK showed enhanced activity in COL hydrogel while with decreased frequency in pulsation. Conclusion ERK has autonomous pulsating activity in Jurkat T cells, which frequency seems regulated by matrix stiffness. The physiological implication of ERK oscillation and underlying molecular mechanism need further study.