No blood-based protein biomarkers are currently available for routine clinical use to determine the prognosis of patients with glioblastoma (GB). We performed data-independent acquisition mass spectrometry (DIA-MS)-based proteomics on 96 presurgical serum samples from patients with GB and 30 serum samples from healthy controls to identify such markers. Among the 622 serum proteins differentially expressed between the GB and control groups, 191 had a |log2(fold change)| ≥ 0.58 and an area under the curve ≥ 0.75. An analysis of their prognostic value revealed that high levels of IL1R2 and low levels of CRTAC1 and HRG were associated with poor survival. Multivariate Cox regression analysis identified IL1R2 as an independent prognostic factor for PFS and CRTAC1 as an independent prognostic factor for OS. The concentration of CRTAC1 in serum samples from an independent cohort of short- and long-term survivors of GB (STS and LTS, respectively) by ELISA was shown to be lower in the STS than in the LTS group. CRTAC1, HRG, and IL1R2 could potentially be used to better inform prognosis and predict treatment response in GB patients