Near-infrared(NIR)lights are powerful tools to conduct deep-tissue imaging since NIR-Ⅰ wavelengths hold less photon absorption and NIR-Ⅱ wavelengths serve low photon scat-tering in the biological tissues compared with visible lights.Two-photon fluorescence lifetime microscopy(2PFLM)can utilize NIR-Ⅱ excitation and NIR-Ⅰ emission at the same time with the assistance of a well-designed fluorescent agent.Aggregation induced emis-sion(AIE)dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA)cross-section as aggre-gated dots.Herein,we report two-photon fluorescence lifetime mi-croscopic imaging with NIR-Ⅱ excitation and NIR-Ⅰ emission using a novel deep-red AIE dye.The AIE-gens held a 2PA cross-section as large as 1.61×104 GM at 1040 nm.Prepared AIE dots had a two-pho-ton fluorescence peak at 790 nm and a stable lifetime of 2.2 ns un-der the excitation of 1040 nm femtosecond laser,The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system.Abundant vessels as small as 3.17 μm were still observed with a nice signal-background ratio at the depth of 750 μm.Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.