Abstract: The error brought up by the sensitivity difference of sensors was analyzed in the transfer function measurement using two-sensor method in acoustic tube. Based on the transfer function found from the known reflectance of known interface the ration of sensors sensitivities could be measured, a new method for sensors sensitivity calibration in acoustic tube was brought forward. An experimental validation of the known transfer function was carried out in air-filled tube with rigid terminator by exchanging the microphones in the acoustic tube. In water-filled tube with air terminator the experimental validations of the new method were carried out in the condition with environment pressure and higher pressure, it illustrates the validity of the new method, which overcomes the difficulty of exchanging hydrophone in water tube.