The present work uses nanoindentation method to measure the hardness of silicone elastomer annealed at 80 °C, 120 °C, 200 °C, 300 °C, and 350 °C for 60 min, respectively. Based on the apparent surface analysis, a linear relationship is found between the hardness and the reciprocal of the penetration depth. It is found that hardness of the polymer sample obviously decreases after annealing at 80 °C, which is due to the removal of any potential surface residual stresses under the initial annealing process. If the annealing temperature is larger than 300 °C, the hardness increased a lot. Therefore, from the characterization of surface hardness, what kind of (delete) temperature ever reached in the device made of silicone elastomer in the biology field can be deduced.