Optical tweezers are widely used to measure the mechanical properties of erythrocytes, which is crucial to the study of pathology and clinical diagnosis of disease. During the measurement, the blood sample is diluted and suspended in an exogenous physiological fluid, which may affect the elastic properties of the cells in vitro. Here, we investigate the effect of different diluents on the elastic properties of mouse erythrocytes by quantitatively evaluating their elastic constants using optical tweezers. The diluents are plasma extracted from mouse blood, veterinary blood diluent (V-52D), Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), and normal saline (NS). To create an environment that closely resembles in vivo conditions, the experiment is performed at 36.5 °C. The results show that the spring constant of mouse erythrocytes in plasma is 6.23 ± 0.41 μN m-1. The elasticity of mouse erythrocytes in V-52D and DMEM is 8.21 ± 0.91 and 6.95 ± 0.85 μN m-1, which are higher than that in plasma extracted from blood, whereas, the elasticity in PBS and NS is 4.23 ± 0.85 and 4.68 ± 0.79 μN m-1, which are less than that in plasma extracted from blood. At last, we observe the size and circularity of erythrocytes in different diluents, and consider that the erythrocyte diameter and circularity may affect cell deformability. Our results provide a reference of the diluent choice for measuring the mechanical properties of erythrocytes in vitro.