Although protein-based biomimetic fibers are available, green and inexpensive protein-based fibers remain a challenge to develop. Herein, a microfluidic chip was designed to prepare protein-based fibers using cellulose nanofibers and various plant-derived proteins, including wheat gluten, zein, soy protein, pea protein, and rice bran protein. Furthermore, a comprehensive elucidation of the plant protein fiber formation mechanism was conducted. The results showed that plant protein fibers prepared by microfluidic spinning had smooth surfaces, strong mechanical properties, high thermal stability and antioxidative activity, good digestibility, and low sensitization. Additionally, the strong hydrodynamic shear force produced during microfluidic spinning changed the secondary structure of the protein, which promoted the binding of protein molecules to cellulose nanofibers, thus enhancing the molecular orientation of both protein and nanofiber. Consequently, this study may facilitate the development of natural, environmentally friendly, and functional protein-based fibers.