Glioblastoma multiforme (GBM) is a common and fatal disease of the central nervous system. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. In the present study, a novel primary glioblastoma cell line was established, named GWH04, from a patient with GBM, and its STR genotype and various tumor parameters were examined. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as the U87 cell line, but a faster rate than the GL15 cell line, and substantial soft agar clone-formation capacity and subcutaneous and intracranial tumorigenic capacity. For drug sensitivity test, half maximal inhibitory concentration assays for multiple drugs were performed in these three cell lines, and GWH04 cells were found to be resistant to temozolomide. Aneuploid karyotype with a median of 84 chromosomes was possessed by GWH04, as well as chromosomal structural abnormalities, such as broken chromosomes, double centromere chromosomes, homogeneous staining regions, and double microbodies. Gene sequencing further revealed the mutational status of genes TP53, PTEN, PDGFRA, ERBB2, BRCA1, NF1, and MLH1 and the promoter region of telomerase reverse transcriptase (C228T) in this cell line. Altogether, these results indicated that GWH04 will be a useful tool for human GBM studies both in vitro and in vivo.