Chondroitin sulfate (CS) is a glycosaminoglycan actively researched for pharmaceutical, nutraceutical and tissue engineering applications. CS extracted from marine animals displays different features from common terrestrial sources, resulting in distinct properties, such as anti-viral and anti-metastatic. Therefore, exploration of undescribed marine species holds potential to expand the possibilities of currently-known CS. Accordingly, we have studied for the first time the production and characterization of CS from blackmouth catshark (Galeus melastomus), a shark species commonly discarded as by-catch. The process of CS purification consists of cartilage hydrolysis with alcalase, followed by two different chemical treatments and ending with membrane purification. All steps were optimized by response surface methodology. According to this, the best conditions for cartilage proteolysis were established at 52.9 °C and pH = 7.31. Subsequent purification by either alkaline treatment or hydroalcoholic alkaline precipitation yielded CS with purities of 81.2%, 82.3% and 97.4% respectively, after 30-kDa membrane separation. The molecular weight of CS obtained ranges 53–66 kDa, depending on the conditions. Sulfation profiles were similar for all materials, with dominant CS-C (GlcA-GalNAc6S) units (55%), followed by 23–24% of CS-A (GlcA-GalNAc4S), a substantial amount (15–16%) of CS-D (GlcA2S-GalNAc6S) and less than 7% of other disulfated and unsulfated disaccharides