The urokinase-type plasminogen activator receptor (uPAR) is overexpressed in several cancers including glioblastoma (GBM) and is an established biomarker for metastatic potential. The uPAR-targeting peptide AE105-NH2(Ac-Asp-Cha-Phe-(D)Ser-(D)Arg-Tyr-Leu-Trp-Ser-CONH2) is a promising candidate for non-invasive positron emission tomography (PET) imaging of uPAR. Despite the optimal physical properties of68Ga for peptide-based PET imaging, low tumor uptakes have previously been reported using68Ga-labeled AE105-NH2-based tracers. In an attempt to optimize the tumor uptake, we developed three novel tracers with alkane (AOC) and polyethylene glycol (PEG) spacers inserted between AE105-NH2and the radio metal chelator 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA). The resulting tracers NODAGA-AOC-AE105-NH2, NODAGA-PEG3-AE105-NH2and NODAGA-PEG8-AE105-NH2were compared to the non-spacer version, NODAGA-AE105-NH2. Following radiolabeling with68Ga, we evaluated the in vitro and in vivo performance in mice bearing subcutaneous tumors derived from the uPAR-expressing human GBM cell line U87MG. In vivo PET/CT imaging showed that introduction of PEG spacers more than doubled the in vivo tumor uptake after 1 h compared with the non-spacer version:68Ga-NODAGA-PEG3-AE105-NH2(2.08 ± 0.37%ID/g) and68Ga-NODAGA-PEG8-AE105-NH2(2.01 ± 0.22%ID/g) vs.68Ga-NODAGA-AE105-NH2(0.70 ± 0.40%ID/g), p < 0.05. In addition,68Ga-NODAGA-PEG8-AE105-NH2showed significantly higher (p < 0.05) tumor-to-background contrast (3.68 ± 0.23) than the other tracers. The specific tumor-targeting property of68Ga-NODAGA-PEG8-AE105-NH2was established by effectively blocking the tumor uptake with co-injection of unlabeled AE105-NH2(1 h: unblocked 2.01 ± 0.22%ID/g vs. blocked 1.24 ± 0.09%ID/g, p < 0.05). Ex vivo biodistribution confirmed the improved tumor uptakes of the PEG-modified tracers.68Ga-NODAGA-PEG8-AE105-NH2is thus a promising candidate for human translation for PET imaging of GBM.