L-Theanine, a non-protein amino acid naturally present in tea leaves, is renowned for its antioxidant, anti-inflammatory, and neuroprotective properties, positioning it as a potent functional food ingredient. This study explores the impact of L-Theanine on the lifespan of Caenorhabditis elegans, a model organism whose metabolic and genetic pathways closely mirror those of humans, making it a platform for evaluating the health benefits and potential risks associated with dietary components. This model is highly utilized in the development and testing of nutritional foods. Through advanced glycation end product (AGE) content analysis, we found that L-Theanine significantly reduced the accumulation of AGEs by 26.5%, which are key biomarkers linked to aging and various age-related diseases. When administered during early adulthood, L-Theanine extended the lifespan of C. elegans by 8.7% under normal conditions and by 7.3% under high-sugar-induced stress conditions. This suggests that L-Theanine not only promotes longevity under standard conditions but also offers protective effects against high-glucose-induced stress. Subsequent gene expression and mutant analyses revealed that the lifespan-extending effects of L-Theanine are mediated through the insulin-like signaling pathway, involving the critical regulators DAF-2 and DAF-16. DAF-2, an insulin-like receptor, and DAF-16, its downstream transcription factor, play an important role in controlling genes associated with aging. Our findings demonstrate that L-Theanine extends lifespan by modulating the DAF-2/DAF-16 signaling pathway and reducing AGE accumulation. Our results highlight tea leaves' and L-Theanine's potential as an anti-aging intervention in functional foods, demonstrating their capability to modulate significant aging-related pathways and offering a foundation for future research into their benefits in mammals, including humans.