Background <p>L-theanine, an amino acid derived from green tea, has been suggested to alleviate affective symptoms and enhance cognitive performance, but its clinical relevance remains uncertain.</p> Methods <p>We conducted a systematic review and meta-analysis of 31 randomised controlled trials (n = 1168) comparing oral L-theanine with placebo in healthy and clinical populations. The primary outcome was the acute effect of a single dose on stress in healthy adults. Secondary outcomes included clinical measures (stress after repeated dosing, anxiety, depressive symptoms, and fatigue) and cognitive measures (reaction times and attention tasks with single and repeated doses). Safety was assessed by comparing dropout rates and reasons between L-theanine and placebo groups.</p> Results <p>A single 200 mg dose taken 30–60 min before cognitive testing significantly improved choice reaction time (Standardised Mean Difference [SMD] = 0.51; 95% CI, 0.25–0.77), indicating enhanced attention. The reduction in acute stress was modest (SMD = 0.31) and largely influenced by studies with a high risk of bias. No significant effect on fatigue was observed. Excluding one outlier, a significant reduction in depressive symptoms following a single dose in healthy individuals was found (SMD = 0.69; 95% CI, 0.13–1.25) with low heterogeneity. Anxiety effects were inconsistent and non-significant except for one study on psychotic anxiety (SMD = 0.54; 400 mg/day for 8 weeks). No serious adverse events were reported.</p> Conclusions <p>L-theanine is safe and shows a robust short-term benefit on attention in healthy adults, with a potential antidepressant effect warranting confirmation in high-quality trials, especially in clinical populations.</p>