This study investigates the utilization of squid pen waste through a biocompatible ionic liquid approach, focusing on choline acetate, [Ch][OAc]. This ionic liquid effectively extracts over 80 wt % of protein from squid pen waste. To optimize the extraction process, a factorial design of experiments was employed to achieve a protein recovery of 75% at an estimated purity of 86%, along with highly acetylated, crystalline β-chitin with a purity of up to 95%. The extracted protein was subsequently used to create biocomposite films from α- and β-chitosan, demonstrating impressive tensile strengths of 93.15 ± 7.9 and 83.5 ± 6.2 MPa, respectively, while maintaining hydrophilic properties (θ