The poor clinical prognosis and microvascular patterns of glioblastoma (GBM) are of serious concern to many clinicians and researchers. However, very few studies have examined the correlation between microvascular niche patterns (MVNPs) and proteomic distribution. In this study, CD34 immunofluorescence staining and matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-IMS) technology were used to investigate the protein distributions in MVNPs. CD34+ microvascular phenotype could be divided into four types: microvascular sprouting (MS), vascular cluster (VC), vascular garland (VG), and glomeruloid vascular proliferation (GVP). Based on such characteristics, MVNPs were divided into two types by cluster analysis, namely, type I, comprising primarily MS and VC, and type II, comprising many VGs and GVPs. Survival analysis indicated the type of MVNPs to be an independent prognostic factor for progression-free and overall survival in GBM. MALDI-IMS results showed the peaks at m/z 1037 and 8960 to exhibit stronger ion signals in type II, while those at m/z 3240 and 3265 exhibited stronger ion signals in type I. The findings may assist future research on therapy and help predict prognosis in GBM. However, due to the limited number of studies, more well-designed studies are warranted to further verify our results.