Dual-color two-photon laser scanning microscopy is a useful method for simultaneously studying the expression, localization and trafficking of two different proteins in tissues. Because most two-photon microscopes only use a single wavelength excitation laser, simultaneously exciting multiple fluorescent proteins remains a challenge. Here, we present mAmetrine and mKate2, which can be used as a novel fluorescent protein pair in dual-color two-photon imaging by taking advantage of the large Stokes shift of mAmetrine and high brightness of mKate2. Both proteins have high two-photon absorption efficiencies and can be simultaneously excited at an optical wavelength of 765 nm. Dual-color two-photon imaging using this protein pair is highly effective in living cells.