Plant protein-based nanofibers generated by eco-friendly waterborne electrospinning are emerging as sustainable and innovative materials with vast applications in different biomedical areas. In this study, we fabricated electrospun nanofibers based on potato, pea, and soy protein isolates, achieving remarkably high protein content without the use of organic solvents, strong bases, or surfactants. The different protein nanofibers were characterized by means of quantitative fluorescence imaging, optical spectroscopy, and dynamic mechanical analysis. Results indicated that the intrinsic nature of the proteins modulated the properties of the nanofibers in terms of morphology, fluorescence fingerprints, mechanical strength, and stability in aqueous environments. Pea and soy protein isolates, both rich in beta-structure, led to the formation of robust and dense nanofibers, which slowly disintegrated in water. On the contrary, less dense and highly soluble nanofibers were generated from the structurally more flexible potato protein isolate, and these nanofibers demonstrated lower resistance to breakage. Our findings indicate the importance of protein structural elements when designing protein-based electrospun nanofibers with specific features. Deciphering the intricate relationship between protein structure at the molecular level and properties of nanofiber holds promise for the development of biomaterials with enhanced efficacy in diverse biomedical applications.