Mass transfer limitation in conventional top-down tissue engineering makes it impossible to fabricate large size viable tissue replacements. In the present study, we aimed at performing a systemic investigation of the assembling process in perfusion culture for fabricating centimeter-scale macrotissues from cell-laden microcarriers following a bottom-up modular approach. Cells (human fibroblasts, human mesenchymal stem cells, or HepG2 cells) were seeded onto microcarriers (Cytopore-2 or CultiSpher S) in spinner flasks and cultured for 14 days and subsequently transferred to a perfusion chamber for assembling. It was found that growth of different cells on different microcarriers varied and aggregation of cell-laden microcarriers was favored with CultiSpher S. After perfusion culture for 14 days, while all microtissues could assemble into integral macrotissues, macrotissues of HepG2 cells were structurally most inferior and the assembling of cell-laden CultiSpher S led to a significant shrinkage. By designing perfusion chamber and using agar-based templates, tubular, disc, and alphabetic letter-shaped macrotissues could be easily fabricated, suggesting template-assisted assembling. Importantly, it was revealed that there existed both optimal perfusion culture time (21 days) and packing condition (1/4 compression) for the assembling of microtissues. This study lays a solid foundation for future applications of this microtissue assembling technique in tissue engineering.