An accurate prediction of the intracranial infiltration tendency and drug response of individual Glioblastoma (GBM) cells is essential for personalized prognosis and treatment for this disease. However, the clinical utility of mouse PDOX models remains limited given current technical constraints including difficulty in generating sufficient sample numbers from small tissue samples and a long latency period for results. To overcome these issues, we establish zebrafish GBM xenografts of diverse origin, which can tolerate intracranial engraftment and maintain their unique histological features. Subsequent single cell RNA-seq (scRNA-seq) analysis confirms significant transcriptional identity resemble invading GBM microtumors observed in the proportionally larger brains of model animals and humans. Endothelial scRNA-seq confirms zebrafish blood-brain-barrier is homologous to the mammalian. Finally, we establish a rapid and efficient zebrafish PDOX (zPDOX) model, which can predict long-term outcomes of GBM patients within 20 days. zPDOX provides a novel avenue for precision medicine of GBM, especially for the evaluation of intracranial infiltration tendency and prediction of individual drug sensitivity.© 2022. Published by The Company of Biologists Ltd.