This study aimed to investigate the protective effect and molecular mechanism of theanine on high-sucrose diet-induced damage in the nematode model Caenorhabditis elegans. We analyzed the changes in the behavior, development, reproductive capacity, oxidative stress levels, and related gene expression in nematodes following high-sucrose diet-induced damage and theanine intervention (at concentrations of 50 to 1000 μg/mL). The results showed that the high-sucrose diet significantly inhibited the growth and development of C. elegans, reduced its reproductive capacity, and exacerbated oxidative stress. Low-dose theanine (50 μg/mL) effectively alleviated high-sucrose diet-induced damage, while higher doses of theanine (500 and 1000 μg/mL) aggravated oxidative damage. Molecular mechanism studies demonstrated that theanine alleviated oxidative damage by activating fat-2, inducing the expression of cat-1 and sod-3, restoring the expression of gst-4, and increasing the expression of polg-1, thereby regulating three pathways: lipid metabolism, antioxidant defense, and mitochondrial function. In summary, theanine exerted a dose-dependent bidirectional regulatory effect on high-sucrose diet-induced damage, either exerting a protective effect at low doses or potentially causing harmful effects at high doses, through the synergistic regulation of multiple pathways. This study provides a theoretical basis for exploring nutritional interventions for diabetes, but its clinical application requires strict control of the safety dose threshold and further validation in mammalian models.