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Aspergillus niger is an important strain for industrial production of organic acids and enzymes, possessing a powerful hydrolytic enzyme system and glucose transport system, which enable to copy with extracellular carbon sources variance and uptake glucose to support cell growth and industrial production. As an important carbon source and an essential signaling molecule, glucose and its uptake has considerable influence on cell growth and fermentation performance. Regarding to issue of lacking simple methods for quantitative characterization of glucose absorptive capacity in filamentous fungi, we developed a quantitative detection pipeline for glucose uptake capacity assay for filamentous fungi, using a non-metabolizable,flourscent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-yl)amino)-2-deoxyglucose(2-nbdg) as a fluorescent probe, then incubating pre‑cultured A. niger spores with 2‑NBDG, and using with fluorescence microscopy and flow cytometry. It showed that the optimized 2‑NBDG work concentration and incubation time were 150 μmol/L and 4 h, respectively. Using this quantitative analysis method, it was found that the overexpression of low affinity glucose transporter MstC increased glucose uptake by 1.44 times. Meanwhile, on the basis of previous studies, mutant R188K of MstC was designed, and the point mutation directly caused the loss of glucose transport activity of MstC, which indicates that Arg188 is a key amino acid site affecting the glucose transport capacity of MstC. The establishment of this quantitative glucose uptake detection method and its application to the study of MstC function not only deepens the quantitative understanding of glucose absorption in filamentous fungi, but also provides technical support for the transformation and optimization of glucose transport system. This study provides a feasible quantitative approach to investigate the glucose uptake of filamentous fungi, and also revealed the physiological function of key glucose transporter MstC, which paves the way for glucose uptake engineering in this industrially important fungal cell factory [ABSTRACT FROM AUTHOR]