Squid (Todarodes pacifica) pen was an excellent source of β-chitin with 25.5% yield. The optimal condition to prepare squid pen β-chitin was established: deproteinisation with 3% NaOH for 30 min at 15 psi/121 °C and a solid/solvent ratio of 1:10 (w/v) and a subsequent demineralisation with 1 N HCl for 30 min at room temperature and a solid/solvent ratio of 1:10 (w/v). Squid pen β-chitin contained 6.29% nitrogen, 0.25% ash, and negligible fat with degree of acetylation of 94.02%, residual amino acid of 0.499 g/100 g and bulk density of 0.28 g mL−1. Depending on its particle size, squid pen β-chitin visually looked white (L* = 82.82, a* = −0.67, b* = 6.31; particle size of 0.15–0.18 mm) or light grey (L* = 62.88, a* = 0.33, b* = 10.66; particle size of 0.425–0.841 mm). Water, fat and dye-binding capacity of squid pen β-chitin was 694.67%, 194.03% and 79.81%, respectively.