Although protein-based fibers have been developed, the preparation of inexpensive, safe and environmentally friendly plant protein fibers is still a hot topic. Herein, we constructed the microfluidic chip to produce protein fiber using pea protein and a small amount of cellulose nanofibers. The results demonstrated that compared to pea protein alone, the pea protein-based fiber produced through microfluidic spinning exhibited a smooth and dense surface structure, good thermal stability, higher Zeta potential and antioxidant activity, and lower surface hydrophobicity and sensitization. Furthermore, we comprehensively revealed the dynamic formation mechanism of pea protein-based fiber. During the fiber preparation process, hydrodynamics led the protein and cellulose nanofibers to align gradually along the flow direction, enhancing their molecular orientation. Additionally, strong hydrodynamic shear force altered the protein structure, transitioning its spatial structure from random coil and α-helix to β-sheet, and promoting a binding of protein to cellulose nanofibers. This research may offer theoretical guidance for the application of pea protein-based fiber in the preparation of hypoallergenic protein-based fibers and the construction of foodborne delivery materials.