The intra- and intermolecular interactions of chitin in NaOH/urea aqueous system were studied by a combination of NMR measurements (including C-13 NMR, Na-23 NMR, and N-15 NMR) and differential scanning calorimetry. The results revealed that the NaOH and chitin formed a hydrogen-bonded complex that was surrounded by the urea hydrates to form a sheath-like structure, leading to the good dissolution. The optimal concentration range, in which chitin was molecularly dispersed in NaOH/urea aqueous, was found to investigate the chain conformation in the dilute solution with a combination of static and dynamic light scattering. The weight-average molecular weight (M-w), radii of gyration (< R-g >(z)), and hydrodynamic radii (< R-h >(z)) values of chitin were determined, and the structure-sensitive parameter (rho) and persistent length (L-p) were calculated to be >2.0 and similar to 30 nm, respectively, suggesting an extended wormlike chain conformation. The visualized images from TEM, cryo-TEM, and AFM indicated that, chitin nanofibers were fabricated from the parallel aggregation of chitin chains in NaOH/urea system. This work would provide a theoretical guidance for constructing novel chitin-based nanomaterials via bottom-up method at the molecular level.