Objective To study 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 the cells were embedded into type I collagen (COL) hydrogel to detect the biomechanical effect. Results Pulsation of ERK activity was found in a sub-group of Jurkat cells, the frequency was about three times per hour, and the average change in oscillating magnitude was about 20%. Under the condition of T-cell receptor (TCR) activation with antibodies, ERK pulse still existed, but the frequency and amplitude did not change significantly. ERK showed a decreased frequency of pulsation in COL hydrogel with an increased stiffness. Conclusions ERK has autonomous pulsating activity in Jurkat T cells, and preliminary experiments show that the frequency is regulated by matrix stiffness. The physiological implication of ERK oscillation and underlying molecular mechanism need further study.