Deformation and rotation process of an interface between two fluids with the same properties has been Studied in a three-dimensionally bent channel, which consisted of upstream and downstream square channels connected via a circular channel. A straight fluid interface parallel to the sidewall of the upstream channel was formed in the central region by introducing two fluids with the same properties at an equi-volume rate from the branches of a Y-shaped channel into its stern, which was used as the upstream channel. The fluid interface thus formed was visualized in the upstream and downstream channel cross-sections by the Laser Induced Fluorescence (LIF) method, and its deformation and rotation were investigated. In the case of Reynolds number larger than 2, the interface was curved and stretched by a secondary flow generated in the flow from upstream to down-stream via the circular channel, whose length increased linearly to the logarithm of Reynolds number. In the case of Reynolds number less than 2, the interface rotated but was deformed a little from its initial straight shape while the fluid passed through the bent channel. The rotation angle of the fluid interface in the exit cross-section perpendicular to the flow direction was almost equal to the crossed axis angle between upstream and down-stream channels. CFD calculations of the position, shape and rotation angle of the fluid interface were in good agreement with the experimental results. The CFD analysis for a rectangular channel demonstrates that the linearized fluid interface rotates almost by the crossed axis angle of a three-dimensionally bent channel, and that the deformation of the fluid interface is small provided that the channel aspect ratio is less than one or the channel height is larger than the channel width.