Actin cytoskeleton reorganization, which is governed by actin-associated proteins, has a close relationship with the change of cell biological behavior. However, a perceived understanding of how actin mechanical property links to cell biological property remains unclear. This paper reports a label-free biomarker to indicate this interrelationship by using the actin cytoskeleton model and optical tweezers (OT) manipulation technology. Both biophysical and biochemical methods were employed, respectively, as stimuli for two case studies. By comparing the mechanical and biological experiment results of the leukemia cells under electrical field exposure and human mesenchymal stem cells (hMSC) under adipogenesis differentiation, we concluded that β-actin can function as an indicator in characterizing the alteration of cellular biological behavior during the change of actin cytoskeleton mechanical property. This study demonstrated an effective way to probe a quantitative understanding of how actin cytoskeleton reorganization reflects the interrelation between cell mechanical property and cell biological behavior.