The blood-brain barrier (BBB) is a limiting factor in nanoparticle drug delivery to the brain, and various attempts have been made to overcome it for efficient drug delivery. Nowadays, it was considered as further issue for brain-drug delivery that the nanoparticle delivered to brain through the BBB reach cancer cells in tumour tissue. In this study, we investigated the effect of nanoparticle size on blood-brain tumour barrier (BBTB) permeation of fluorescence-labelled gold nanoparticles (AuNPs) in a mouse model of orthotopic glioblastoma multiforme (GBM), established by intracranial implantation of luciferase-expressing human glioblastoma U87MG cells. AuNPs sized 10, 50, and 100 nm were perfused into the GBM mice via internal carotid artery (ICA) for 5 min. Immediately after perfusion, the brains were fixed and prepared for LSCM observation. The AuNPs distribution in the normal and tumorous brain tissues was analysed qualitatively and quantitatively. Higher distribution of AuNPs was observed in the tumorous tissue than in the normal tissue. Furthermore, the smallest nanoparticle, 10 nm AuNPs, was widely distributed in the brain tumour tissue, whereas the 50 and 100 nm AuNPs were located near the blood vessels. Therefore, nanoparticle size affected the permeation of nanoparticles from the blood into brain tumour tissue.