Objective: Surgical eradication of malignant glioma cells is theoretically impossible. Therefore, reducing the number of remaining tumor cells around the brain-tumor interface (BTI) is crucial for achieving satisfactory clinical results. The usefulness of fluorescence-guided resection for the treatment of malignant glioma was recently reported, but the detection of infiltrating tumor cells in the BTI using a surgical microscope is not realistic. Therefore, we developed an intraoperative rapid fluorescence cytology system, and evaluated its clinical feasibility for the management of malignant glioma. Materials and methods: Twenty-five selected patients with malignant glioma (newly diagnosed: 17; recurrent: 8) underwent surgical resection under photodiagnosis using photosensitizer Talaporfin sodium and a semiconductor laser. Intraoperatively, a crush smear preparation was made from a tiny amount of tumor tissue, and the fluorescence emitted upon 620/660 nm excitation was evaluated rapidly using a compact fluorescence microscope in the operating theater. Results: Fluorescence intensities of tumor tissues measured using a surgical microscope correlated with the tumor cell densities of tissues evaluated by measuring the red fluorescence emitted from the cytoplasm of tumor cells using a fluorescence microscope. A “weak fluorescence” indicated a reduction in the tumor cell density, whereas “no fluorescence” did not indicate the complete eradication of the tumor cells, but indicated that few tumor cells were emitting fluorescence.Conclusion: The rapid intraoperative detection of fluorescence from glioma cells using a compact fluorescence microscope was a useful to evaluate the presence of tumor cells in the resection cavity walls, and provides surgical implications for the more complete resection of malignant gliomas.