Chondroitin sulphate (ChS) from the scapular cartilage of the shortfin mako shark (Isurus oxyrinchus) was purified by two-stage enzymatic hydrolysis and a fractional precipitation process using isopropyl alcohol. Characteristics of the ChS fraction were investigated using cellulose acetate membrane electrophoresis and FT-IR spectra. A maximum hydrolysis rate of 78.26% was achieved with 1.35% (w/w) Alcalase and 1.20% (w/w) Flavourzyme. A minimum nitrogen content of 2.89% was obtained with 1.43% (w/w) Alcalase and 1.33% (w/w) Flavourzyme, as determined by response surface methodology. The precipitation of ChS from the enzymatic hydrolysates was optimised at 40% (v/v) isopropyl alcohol, which contained 2% (w/v) NaCl to lower the nitrogen content. The precipitate was further purified via membrane filtration (molecular-weight cut-off, 3 kDa) to remove salt and low-molecular-weight materials. The ChS purified by enzymatic hydrolysis, isopropyl alcohol precipitation and membrane filtration was identified as ChS C by electrophoresis and FT-IR spectra.