2,2,6,6-Tetramethylpiperidin-1-yl oxyl (TEMPO)-oxidized beta-chitin nanofibrils (T-ChNF) are novel nanofibrils of high strength and stiffness and can also enhance the adsorption of chitosan in materials for biomedical applications. This study presents the preparation, structure, and properties of T-ChNF based on squid pens. Our nanofibrils have a zeta potential of -25.3 mV at neutral pH and a carboxylic content of 0.17 mmol/g, making hydrocolloid suspension stable at alkaline and neutral pH. It was demonstrated that positively charged chitosan could be adsorbed on the negatively charged surface of T-ChNF, leading to charge neutralization. The key to strong squid pen T-ChNF is the initial raw squid chitin properties and the high degree of acetylation (DA = 99.9%). The T-ChNF diameter is similar to 4.4 nm, and the length is in the micrometer range. The length and diameter are similar to those of squid pen beta-chitin fibrils prepared under mild conditions. These qualities of fibrils resulted in high-strength (176 MPa) chitin films prepared using the rapid vacuum filtration and drying technique. T-ChNF-based films are ductile, flexible, and transparent.