Glioblastoma　(GBM)　is　a　highly　angiogenesis　malignant　tumor　with　a　significant　heterogeneity　of　vascular　morphology.　The　vascular　patterns　could　be　served　as　an　indicator　of　vascular　morphological　complexity　that　associated　with　postoperative　prognosis　of　patients　with　GBM.　Here,　multiphoton　microscopy　(MPM),　based　on　second　harmonic　generation　(SHG)　and　two-photon　excited　fluorescence　(TPEF),　is　applied　for　detecting　the　architectural　feature　of　blood　vessels　from　ex　vivo,　unfixed,　and　unstained　human　GBM　specimens.　Combined　with　the　image　processing　algorithms,　the　morphology,　location,　orientation,　alignment,　and　density　of　vascular　collagen　are　qualitatively　and　quantitatively　visualized,　revealing　the　presented　method　not　only　contribute　to　differentiate　GBM　from　normal　brain　tissue,　but　also　provide　important　indicators　of　different　vascular　patterns,　which　may　serve　as　a　reference　to　the　prognosis　more　accurately.　Therefore,　these　results　can　provide　prognosis-related　histopathological　diagnosis　of　GBM,　holding　a　translational　potential　to　be　used　as　a　label-free,　quick,　and　on-site　imaging　tool　for　clinicians　to　determine　the　extent　of　excision　involving　brain　functional　areas.