Abstract: A study site was selected within a mid-mountain moist evergreen broad-leaved forest plot located in the core area of the Ailao Mountains National Nature Reserve in Yunnan Province, 30 permanent 20 m × 20 m forest plots were established. The study used allometric growth equations to estimate the aboveground biomass (AGB) of woody plants, and applied partial least squares (PLS) regression to assess the relative influence of environmental factors, functional diversity, and phylogenetic diversity on the AGB of the overstorey and understory tree layers. The results showed that the AGB of the overstorey and understorey tree layers was 346.28 ± 137.35 t/hm² and 12.35 ± 8.73 t/hm², respectively, with the PLS models explaining 40.7% and 63.1% of their variation. Compared with functional traits and phylogenetic diversity, environmental factors played a more significant role in shaping overstorey AGB, and the strong correlation between overstorey AGB and community-weighted mean trait values supported the "Mass Ratio Hypothesis". Understorey AGB exhibited a stronger correlation with community-weighted traits and Functional Richness (FRic) than with environmental factors, supporting both the "Mass Ratio Hypothesis" and the "Niche Complementarity Hypothesis". Overstorey AGB was positively correlated with the phylogenetic diversity indices Faith's PD and Mean Pairwise Distance (MPD), while understorey AGB was positively correlated with Faith's PD but negatively correlated with the Mean Nearest Taxon Distance (MNTD), indicating that phylogenetic diversity exerts distinct influences on AGB accumulation between the overstorey and understorey layers. The study elucidated the primary factors affecting the AGB of both the overstorey and understorey tree strata within the mid-mountain moist evergreen broad-leaved forests of the Ailao Mountains, thereby offering insights for research into the sustaining mechanisms of biomass carbon pools in subtropical forest ecosystems.