Glycans, unlike uniformly charged DNA and compositionally diverse peptides, are typically uncharged and possess rich stereoisomeric diversity in the glycosidic bonds between two monosaccharide units. These unique features, including charge heterogeneity and structural complexity, pose significant challenges for accurate analysis. Herein, we developed a novel single-molecule oligosaccharide sensor, OmpF nanopore. The natural electroosmotic flow within OmpF generates a robust driving force for unlabeled neutral oligosaccharides, enabling detection at a concentration as low as 6.4 μM. Furthermore, the asymmetric constriction zone of OmpF was employed to construct a stereoselective recognition site, enabling sensitive identification of glycosidic bond differences in cell lysate samples. With the assistance of machine learning algorithms, the OmpF nanopore achieved a recognition accuracy of 99.9 % for tetrasaccharides differing in only one glycosidic bond was achieved. This nanopore sensor provides a highly sensitive analytical tool with a broad dynamic range. It enables chiral recognition of oligosaccharides at low concentrations and is suitable for analysing both low-abundance and practical samples.