<p>Chitin, distinguished by its nitrogen-rich acetamido and amino groups, imparts a distinctive cationic nature, enabling chitin to have indispensable features in various applications. Despite its significant promise in the textile industry, particularly for sustainable and functional fabric applications, the practical utilization of chitin fibers remains constrained by insufficient mechanical strength. The degree of deacetylation (DD), a key molecular-level structural determinant, has not been adequately addressed in previous studies despite its critical role in influencing chitin properties across multiple scales. In this study, a deacetylation-mediated design strategy was used to achieve enhanced mechanical performance coupled with multifunctional efficacy using an aqueous KOH/urea solution dissolution system. We prepared a series of deacetylated chitins with different DD values and systematically studied the effect of deacetylation on the multiple-scale structure of regenerated fibers, such as intermolecular interactions and chain orientation at the molecular level, and the aggregation behavior of chitin nanofibers within the gel-state and dried fibers at the micro/nano scale. To achieve an enhanced mechanical performance coupled with multifunctional efficacy by relying on an aqueous KOH/urea solution dissolution system. Moreover, deacetylation enhances intermolecular interactions, resulting in densified internal structures and improved fiber orientation. Concomitantly, it augmented the antimicrobial functionality of the fibers. This deacetylation-mediated design strategy provides a deeper understanding of the structure and properties of regenerated chitin and advances the utility of chitin in strong and sustainable fibers.</p>