Numerous studies have shown that type II collagen (CII) has a potential role in the treatment of rheumatoid arthritis. However, most of the current studies have used terrestrial animal cartilage as a source of CII extraction, with fewer studies involving marine organisms. Based on this background, collagen (BSCII) was isolated from blue shark (Prionace glauca) cartilage by pepsin hydrolysis and its biochemical properties including protein pattern, total sugar content, microstructure, amino acid composition, spectral characteristics and thermal stability were further investigated in the present study. The SDS-PAGE results confirmed the typical characteristic of CII, comprising three identical α1chains and its dimeric β chain. BSCII had the fibrous microstructure typical of collagen and an amino acid composition represented by high glycine content. BSCII had the typical UV and FTIR spectral characteristics of collagen. Further analysis revealed that BSCII had a high purity, while its secondary structure comprised 26.98% of β-sheet, 35.60% of β-turn, 37.41% of the random coil and no α-helix. CD spectra showed the triple helical structure of BSCII. The total sugar content, denaturation temperature and melting temperature of BSCII were (4.20 ± 0.03)%, 42 °C and 49 °C, respectively. SEM and AFM images confirmed a fibrillar and porous structure of collagen and denser fibrous bundles formed at higher concentrations. Overall, CII was successfully extracted from blue shark cartilage in the present study, and its molecular structure was intact. Therefore, blue shark cartilage could serve as a potential source for CII extraction with applications in biomedicine.