Accumulating evidence suggests that a subpopulation of stem-cell-like tumor cells in glioma (GSCs) is the major factor accounting for intratumoral heterogeneity and acquired chemotherapeutic resistance. Therefore, understanding intratumoral heterogeneity of GSCs may help develop more effective treatments against this malignancy. However, the study of GSCs’ heterogeneity is highly challenging because tumor stem cells are rare. To overcome the limitation, we employed a microfluidic single-cell culture approach to expand GSCs by taking advantage of the self-renewal property of stem cells. Stemness of the recovered cells was confirmed by immunofluorescence, RT-PCR, RNA-sequencing, and cell function assays. The recovered cells were classified into three groups based on their morphological characteristics, namely, the tight-format (TF), the loose-format (LF), and the limited-size group (LS). The serial passage assay showed that the LS group has a lower sphere-forming rate than the LF and TF group, and the invasion assay showed that the LF and TF cells migrated longer distances in Matrigel. The transcriptomic analysis also revealed differences in gene expression profiling among these GSC subtypes. The abovementioned results suggest that GSCs have transcriptional and functional heterogeneities that correlate with morphological differences. The presented microfluidic single-cell approach links morphology with function and thus can provide an enabling tool for studying tumor heterogeneity.