Abstract Current brain organoid technology fails to provide adequate patterning cues to induce a mature structure that represent the complexity of the human brain. Here, we developed a module-based cellular reconstitution technology to sequentially build uniform forebrain assembloids with mature cortical structures and functional connectivity. The uniformity and maturity of the newly-conceived forebrain assembloids were 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 forebrain assembloids were 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 forebrain assembloids structurally and functionally recapitulated 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 provided 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.