Posidonia oceanica (L.) Delile is a seagrass endemic to the Mediterranean Sea and a significant contributor to human well-being through a variety of ecosystem functions, such as carbon cycling. Despite its ecological significance, most methods for estimating leaf biomass in this species are either destructive or expensive. In this study, 2500 individual leaves from 351 shoots of Posidonia oceanica were collected, across 16 sites in the Ligurian Sea, over two time periods (2016–2018 and 2024–2025), and analyzed for total leaf area and dry weight. An allometric equation following a power-law structure was derived using linear mixed-effects models and it was later validated via 10-fold cross-validation. Although some variations in the intercept term were observed, the allometric scaling structure remained consistent across space and time, providing the first robust species-specific allometric tool to estimate P. oceanica biomass, forming the basis of a proposed non-destructive protocol for meadow-scale biomass estimation.