An otitis media with effusion model in human temporal bones with two laser vibrometers was created in this study. By measuring the displacement of the stapes from the medial side of the footplate, the transfer function of the middle ear, which is defined as the displacement transmission ratio (DTR) of the tympanic membrane to footplate, was derived under different middle ear pressure and fluid in the cavity with a correction factor for cochlear load. The results suggest that the DTR increases with increasing frequency up to 4k Hz when the middle ear pressure was changing from 0 to 20 or −20 cm H2O (e.g., ±196 daPa) and fluid level was increasing from 0 to a full middle ear cavity. The positive and negative pressures show different effects on the DTR. The effect of fluid on DTR varies between three frequency ranges: f<1k, between 1k and 4k, and f>4k Hz. These findings show how the efficiency of the middle ear system for sound transmission changes during the presence of fluid in the cavity and variations of middle ear pressure.