<p>Despite recent advances, current brain organoid technologies face ongoing challenges in managing heterogeneity and representing the diverse structure and cell types of the human brain. Here, we develop a module-based cellular reconstitution technology to sequentially build uniform cortical assembloids with mature cortical structures and functional connectivity. The uniformity and maturity of the cortical assembloids are achieved by creating single-rosette-based organoids at the early stage, whose sizes were big and consistent with the treatment of Wnt and Hedgehog agonists, followed by spatial reconstitution with the Reelin-expressing neuronal layer and non-neuronal glial cells. The resulting single-rosette-based cortical assembloids are highly uniform and reproducible without significant batch effects, solving major heterogeneity issues caused by difficulties in controlling the number and size of rosettes in conventional multi-rosette organoids. Furthermore, these cortical assembloids structurally and functionally recapitulate the physiology of the human brain, including the six-layered cortical structure, functional connectivity, and dynamic cellular interplay between neurons and glial cells. Our study thus provides an innovative preclinical model to study a range of neurological disorders, understanding the pathogenesis of which requires an organoid system capable of representing the dynamic cellular interactions and the maturity of the human brain.</p>