The invention provides a dual-color two-photon fluorescence imaging method and a device, which uses six dual-color beams for emitting along plus and minus X, plus and minus Y, plus and minus Z six directions into the sample 3, thereby forming a three-dimensional lattice for exciting fluorescence, and uses a two-dimension small hole array 6 for filtering background fluorescence in the detecting light path, uses femtosecond pulsed laser with two different kinds of frequencies for composing a dual-color beam, wherein the two are coaxial, and the wavelengths are lambada 1 and lambada 2 respectively, moreover, lambada 1 is equal to lambada 2. Two kinds of light with different wavelength form two different three-dimensional lattice structures in the sample 3, and delay line 10 is modulated for causing the six beams to have the same optical path, only in the zone where two kinds of three-dimensional lattices coincide mutually, the dual-color two-photon fluorescence can be excitated. The device provided by the invention can effectively improve resolution response and imaging speed of three-dimensional imaging, and can be conveniently operated.