Objective: The near-infrared window II (NIRII, 1000–1700 nm) imaging, including NIR-IIa (1300–1400 mm) and NIR-IIb (1500-1700 mm), outperforms the nearinfrared window I (NIR-I, 700-900 nm) imaging in biological researches. However, the advantages of NIR-IIa/IIb imaging in human study are ambiguous. This study aims to apply the NIR-IIa/IIb imaging to glioma resection and evaluate their performance by using the developed imaging instrument and intraoperative image fusion method. Methods: A multispectral fluorescence imaging instrument that integrated NIR-I/II/IIa/IIb fluorescence imaging and an intraoperative image fusion method have been developed. Seven patients with grade III/IV glioma have been enrolled. NIRI/II images of the tumor and NIR-I/II/IIa/IIb images of cerebral vessels were acquired with the administration of indocyanine green. Images were fused using the specialized fusion method to synchronously provide the distribution of the vessels and the surgical boundaries. Results: The NIR-IIa/IIb imaging was successfully applied to the clinic. High imaging resolution and contrast have been attained in the NIR-IIa/IIb spectra. Besides, capillaries with an apparent diameter as small as 182 µm were acquired using NIRIIb imaging. Tumor-feeding arteries were precisely blocked and tumors were excised to the maximum extent for all patients. The blood loss volume during surgery was significantly reduced compared with the control group. Conclusion: The multispectral fluorescence imaging showed high performance, which led to a significant reduction in blood loss volume. Significance: The novel multispectral fluorescence imaging technology can assist surgeons in other vascular surgeries in the future.