Motion of internal wave in three-layer stratified fluid is analyzed by small amplitude wave theory in this paper. Patterns of vertical distribution in velocity due to the wave and its dispersion relationship are discovered, and they fully agree with some characteristics of the internal wave obsered at the field of Osaka Bay. Also, the analyzed results show that velocity of flow is related to rate of discharge in each layer due to the wave, and depth of maximum amplitude depends on state of stratification. Morever, it is found that there are two dilferent modes of velocity in three-layer stratified fluid, interfacial phase and direction of the wave with smaller mode are contrary to case of two-layer stratified fluid. These characteristics of the internal wave in three-layer stratified fluid will be helpful to estimate water environments and to select sultable underwater depth for facilities of aquaculture.