Hypothesis The amount and viscosity of middle ear fluid probably influences the vibration function of the tympanic membrane (TM). Background There has been much research into the mechanisms of hearing loss resulting from middle ear fluid in the previous study. However, further study is required to understand how the middle ear fluid affects the vibration function of the TM. Methods Tests on a TM in a fresh cadaveric human temporal bone specimen under different simulated conditions were carried out. Saline (1 cSt) and silicone oil (100 cSt, 1 000 cSt, 12 500 cSt) were used to simulate middle ear fluid. The fluid approximately contacted 50% or 100% of TM, which was proportional to the fluid amount. Induced by stimulus signal with frequency domain from 0.25 to 8 kHz, the vibration at 6 points of the TM was measured by laser Doppler vibrometer (LDV), respectively. Data acquisition and processing were accomplished by self-developed software. Results With the increase of the fluid amount, the magnitude of velocity transfer function reduced across all frequencies. The effect of fluid viscosity on the TM vibration varied and reduced in a complicated way when the fluid viscosity changed. Conclusion Increasing fluid amount will significantly reduce the TM movement. The effect of fluid viscosity on the TM vibration was nonlinear and related to the fluid amount. The vibration at each point on the TM is frequency-dependent.