Glioma stem cells (GSCs), a particular group of cells from gliomas, are capable of infinite proliferation and differentiation. Recent studies have shown that GSCs may be the root of tumor recurrence, metastasis, and resistance. Early detection and targeted therapy of GSCs may significantly improve the survival rate of glioma patients. Therefore, molecular ligands capable of selectively recognizing GCSs are of great importance. The objective of this study is to generate DNA aptamers for selective identification of the molecular signature of GSCs using cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). GSCs were used as the positive selection target, while U87 cells were used in negative cycles for removal of DNA molecules binding to common glioma cell lines. Finally, we successfully identified one aptamer named W5-7 with a Kd value of 4.9 ± 1.4 nM. The sequence of the aptamer was further optimized, and its binding target was identified as a membrane protein. The aptamer W5-7 was stable in cerebral spinal fluid over 36 h and could also effectively detect glioma stem cells in cerebral spinal fluid samples. With its superb targeting properties and functional versatility, W5-7 holds great potential for use as a molecular probe for detecting and isolating GSCs.