Fatty acid signatures (FA signatures), defined as the relative distribution of saturated, monounsaturated, and polyunsaturated fatty acids within specific biological compartments, have emerged as integrated indicators of dietary lipid exposure, endogenous metabolism, and tissue-specific lipid remodeling. Rather than serving solely as biomarkers of nutritional intake, FA signatures influence membrane architecture, cellular signaling, inflammatory regulation, mitochondrial function, and gene expression, linking dietary lipid quality with metabolic health, chronic disease susceptibility, and biological aging. This review critically synthesized current evidence on the biosynthesis, regulation, analytical assessment, and biological significance of FA signatures, with particular emphasis on cardiometabolic health, neuroinflammation, cognitive aging, and healthy longevity. We distinguish the complementary roles of conventional fatty acid profiling and high-resolution lipidomics in resolving fatty acid composition and molecular lipid species, while highlighting persistent analytical challenges, including biological matrix specificity and the limited resolution of positional and geometric fatty acid isomers. We further examine how integration of lipidomics with nutrigenomics, metabolomics, foodomics, and emerging computational approaches can improve mechanistic interpretation, biomarker discovery, and precision nutrition. Collectively, FA signatures represent dynamic metabolic phenotypes that connect dietary exposure with membrane biology, metabolic regulation, and healthy aging, while providing a promising foundation for evidence-based nutritional strategies and next-generation functional food research.