This work proposes an experimental method for direct measurement of transfer functions of acoustic tubes. The method obtains a pressure-to-velocity transfer function from measurement of input volume velocity and output pressures of a target tube. Steady sinusoidal waves from 100 Hz to 5 kHz with a 10-Hz increment were used as a source signal. Experimental results compared with transmission line simulations indicate the following: (1) transfer functions obtained from the measurements agree well with those from transmission line simulations; (2) differences between the resonant frequencies obtained from the measurements and simulations with a uniform tube are less than 2.6%. These results show conclusive evidence that the proposed method permits accurate measurements of transfer functions of acoustic tubes.