The embodiment of the invention provides a fluorescence collection device, a miniature two-photon microscope and a dual photon imaging method. The fluorescence collection device comprises a first optical path and a second optical path; the first optical path sequentially comprises a first lens, a first optical filter, a dichroic mirror, an infrared filter and a coupling lens; the second optical path sequentially comprises the coupling lens, the infrared filter, the dichroic mirror, a second optical filter and a second lens. According to the fluorescence collection device, the miniature two-photon microscope and the dual photon imaging method, two light pathes are integrated, by selecting filters and dichroic mirrors, reusing infrared filters and coupling lenses, optogenetics controlling and the fluorescence signal collection can be carried out simultaneously in a brain of a free and active animal without increase of weight and volume of the miniature two-photon microscopic probe, different forms of photogenetics stimulation can be realized so as to achieve synchronous collection of fluorescence signals under different optogenetics stimulation, operation is simple, and use is convenient.