Glioblastoma (GBM) is a common malignant tumor of the central nervous system with a poor prognosis and a short survival period. A novel tumor oncolytic virus, Ad-TD-nsIL-12, has manifested anti-tumor properties in preclinical studies. However, the genetic changes caused by Ad-TD-nsIL-12 after GBM treatment are unclear. Therefore, we collected cerebrospinal fluid and tumor tissues from patients injected with Ad-TD-nsIL-12 at different time points and analyzed the methylation and expression profiles of cerebrospinal fluid-derived circulating tumor DNA (ctDNA). The differential genes were screened using the least absolute selection and shrinkage operator (LASSO) and Cox regression analyses. The CIBERSORT algorithm was used to assess the abundance of glioma immune cell infiltration in The Cancer Genome Atlas (TCGA) dataset. The role of hub genes in the diagnosis, prognosis, and immune cell correlation was analyzed using R software, SPSS software, and GraphPad Prism. The results showed that after Ad-TD-nsIL-12 injection, 3631 differential methylation regions (DMRs) were up-regulated and 497 DMRs were down-regulated. The methylation levels of these DMRs recovered within 70 to 82 days. Combined with the TCGA dataset, 8 key genes were selected for the construction of diagnostic and prognostic models. There was a significant correlation between core genes and immune cells. The results revealed that the hub genes in CSF could be used as a biomarker for the diagnosis and prognosis of GBM and led us to speculate the effect of the hub gene on the immune mechanism underlying Ad-TD-nsIL-12.