In order to measure the influence of extracellular Ca2+ concentration on the mechanical properties of human red blood cells, a measurement method of cell membrane shear modulus based on optical tweezers is proposed. An optical tweezers system is integrated on the inverted microscope, the optical tweezers system can utilize the dual traps to transversely stretch and manipulate the human red blood cell attached with microspheres, and analyze the relationship between the cell deformation and the optical trap force, and then the shear modulus of the cell membrane is measured. Through measuring the shear modulus of the red blood cell membrane in the CaCl2 solution with different concentrations, the influence of extracellular Ca2+ concentration on the mechanical properties of red blood cells is analyzed. The results show that compared with normal red blood cells in PBS, the mean shear modulus of red blood cell membrane treated with CaCl2 solutions with concentrations of 100, 200, 300, 400 and 500 μM decreases by 78%, 203%, 344%, 484% and 593%, respectively. In order to verify the measurement results, a mechanical model of red blood cell is established and a uniaxial stretching force is applied to simulate the uniaxial stretching force applied to the cell. The simulation results are consistent with the measured results. This method provides a reference for the measurement of other cellular mechanical properties.