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The cross section is a significant parameter for fluorescence protein and determination of acceptor-to-donor cross section ratio for two-photon excitation in living cells is the vital issue for two-photon excitation FRET quantification. In this study, Hela cells were labeled with FPs that acceptor-to-donor concentration ratio is 1 to 1 and acceptor-to-donor cross section ratio ranged from 700nm to 960nm was obtained by emission spectral unmixing with independent excitation crosstalk correction. The results show that acceptor-to-donor cross section ratio declines with the excitation wavelength from 700nm to 790nm and then increases inversely from 790nm to 960nm. This method can quickly determine the cross section without any additional references, which could provide a powerful and convenient tool for measuring acceptor-to-donor cross section ratio by two-photon excitation in living cells.