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It is of great significance to study the brain structure and function in deep-tissue for neuroscience research and bio-medical applications because of the urgent demand for precise theranostics.Three-photon fluorescence microscopic (3PFM) bioimaging excited by the light in near-infrared lib (NIR-Ⅱb,1,500-1,700 nm) spectral region is one of the most promising imaging techniques with the advantages of high spatial resolution,large imaging depth,and reduced scattering.Herein,a type of NIR-Ⅱb light excitable deep-red emissive semiconducting polymer dots (P-dots) with bright 3PF and large three-photon absorption cross-section (σ3) at 1,550 nm was prepared.Then the P-dots were functionalized with polystyrene polymer polystyrene graft ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH) and modified with NH2-poly(ethylene glycol) (PEG) to synthesis photochemically stable and biocompatible P-dots nanoparticles (NPs).Further the P-dots NPs were utilized for in vivo 3PFM bioimaging of cerebral vasculature with and without the brain skull under 1,550 nm femtosecond (fs) laser excitation./n vivo 3PFM bioimaging of the mice cerebral vasculature at various vertical depths was obtained.Moreover,a vivid three-dimensional structure of the mice vascular architecture beneath the skull was reconstructed.At the depth of 350 μm beneath the brain skull,3.8 μm blood vessels could still be clearly recognized.NIR-Ⅱb excitable P-dots assisted 3PFM bioimaging has great potential in accurate deep tissue bioimaging.