Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are essential functional high-value fatty acids, comprising α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Traditionally, ω-3 PUFAs were primarily obtained from plant oils and marine fatty fish in the diet. Recent advancements in microalgae, filamentous fungi, and oleaginous yeast cultivation have significantly improved the problem of ω-3 PUFA supply shortus and quality unstable, and widely expanded its application scope. ω-3 PUFAs exhibit beneficial effects in preventing cardiovascular disease, neurodegenerative disorders, depression, cancer, promoting infant neurological development, wound healing, and tissue repair. Their molecular mechanisms involve alleviating neurological and systemic inflammation, maintaining cell structure and function, and activating body immune signals. The structure, in vivo digestion, and absorption of ω-3 PUFAs were comprehensively examined. The molecular mechanisms underlying the health benefits of DHA and EPA and potential differences between these fatty acids were systematic analyzed. Additionally, the diverse current applications of ω-3 PUFAs in food products were summerized. Finally, future research directions to optimize ω-3 PUFA production and utilization were prospected. To expand the application of ω-3 PUFAs in food and dietary supplements, interdisciplinary research focusing on improving microbial fermentation technology, constructing efficient gene editing tools, and developing activity maintenance technologies were proposed.