Chitin is one of the most studied biopolymers but the understanding of how it assembles from molecules to microfibers is still limited. Organisms are able to assemble chitin with precise control over polymorphism, texture, and final morphology. The produced hierarchical structure leads to materials with outstanding mechanical properties. In this study, the self-assembly in aqueous solutions of β-chitin nanofibrils, as far as possible similar to their native state, is investigated. These nanofibrils increase their tendency to self-assemble in fibers, up to millimetric length and ≈10 μm thickness, with the pH increasing from 3 to 8, forming loosely organized bundles as observed using cryo-transmission electron microscopy. The knowledge from this study contributes to the understanding of the self-assembly process that follows chitin once extruded from cells in living organisms. Moreover, it describes a model system which can be used to investigate how other biomolecules can affect the self-assembly of chitin nanofibrils.