The most prevalent primary brain tumors are gliomas, which start in the glial cells. Although there have been significant technological advances in surgery and radio-chemotherapy, the prognosis and survival of patients with malignant gliomas remain poor. For routine diagnosis of glioma, computed tomography and magnetic resonance imaging primarily depend on anatomical changes and fail to detect the cellular changes that occur early in the development of malignant gliomas. Therefore, it is urgent to find effective molecular diagnostic tools to detect early stage of malignant gliomas. Currently, cell based-Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX) technology is one effective tool to obtain DNA or RNA aptamers capable of differentiating the molecular signatures among different types of cell lines. Using cell-SELEX, we generated and characterized an aptamer, termed S6-1b, that can distinguish the molecular differences between glioma cell line SHG44 and human astrocytes. Under the conditions of 4 °C and 37 °C, respectively, the dissociation constants of aptamer-cell interaction were both measured in the low nanomolar range. The aptamer S6-1b also exhibited excellent selectivity, making it suitable for use in a complex biological environment. Furthermore, the aptamer can effectively target glioma cells for in vivo fluorescence imaging of tumors. The target type of aptamer S6-1b was identified as a cell membrane protein. Our work indicates that aptamer S6-1b has diagnostic and therapeutic potential to specifically deliver imaging or therapeutic agents to malignant gliomas.