Transcription factor (TF) and microRNA (miRNA) are two best characterized gene regulators that have been found to play an important role in gene regulation. However, high throughput screening the interaction relationships between transcription factors, microRNAs, and target genes in gliomas remains rare. Using GSE16666 and GSE13091 datasets downloaded from Gene Expression Omnibus data, we first screened the differentially expressed genes in gliomas. We explored the regulation relationship among TFs, miRNAs and target genes by different algorithms. The underlying molecular mechanisms of these crucial target genes were investigated by Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Our study has developed three regulation relationships between two TFs and three miRNAs, including TP53/hsa-mir-155, TP53/hsa-mir-125b, and KLF2/hsa-mir-126. In addition, we also constructed a regulation network of the target genes by transcription factors and miRNAs. Some of them had been demonstrated to be involved in glioma progression via various pathways. For example, ATP2B2 target gene could be regulated by has-mir-181a to involve in calcium signaling pathway. RB1 could be regulated by has-miR-26a to participate in pathways in cancer. Smad7 could be regulated by has-miR-21 via intracellular TGF-β signal transduction. We constructed a comprehensive regulatory network which was found to play an important role in gliomas progression.