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Bioactive substances exhibit various physiological activities—such as antimicrobial, antioxidant, and anticancer activities—and have great potential for application in food, pharmaceuticals, and nutraceuticals. However, the low solubility, chemical instability, and low bioavailability of bioactive substances limit their application in the food industry. Using nanotechnology to prepare protein nanoparticles to encapsulate and deliver active substances is a promising approach due to the abundance, biocompatibility, and biodegradability of proteins. Common protein-based nanocarriers include nano-emulsions, nano-gels, nanoparticles, and nano complexes. In this review, we give an overview of protein-based nanoparticle fabrication methods, highlighting their pros and cons. Additionally, we discuss the applications and current issues regarding the utilization of protein-based nanoparticles in the food industry. Finally, we provide perspectives on future development directions, with a focus on classifying bioactive substances and their functional properties.