Trebouxiophyceae, with its morphological and ecological diversity, is the ideal candidate for tracing the origin and evolution of green algae, yet its phylogeny and diversification timeline remain unresolved. Our high-density taxa and large-scale nuclear gene sampling confirm its monophyly, dividing it into the core trebouxiophyceans clade and the Chlorellales clade. We find that incomplete lineage sorting is a possible explanation for nuclear-nuclear discordance. Ancestral state reconstruction suggests that the ancestor of Trebouxiophyceae is likely a unicellular freshwater alga with vegetative reproduction. Furthermore, four calibration strategies infer the origin of Trebouxiophyceae in the late Mesoproterozoic to early Neoproterozoic. The early divergence and diversification of Trebouxiophyceae imply a potential geobiological role in changes in the ancient environments during the Mesoproterozoic-Neoproterozoic transition. This study enhances our understanding of the evolutionary history of green algae and provides insights into the diversification of ancient green plant lineages.