Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated molecules accumulated into the arrival compartment using high-resolution mass spectrometry. Anionic oligosaccharides of various polymerization degrees were discriminated through measurement of the dwelling time and translocation frequency. This molecular sizing capability of the protein nanopore device allowed the real-time recording of the enzymatic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymatically produced oligosaccharides was carried out to monitor the depolymerization enzyme reaction at the single-molecule level.