As a core discipline for the engineering modification of biological systems, synthetic biology is currently confronted with key challenges such as poor functional stability of synthetic communities, insufficient metabolic pathway adaptability of chassis strains, and difficulties in simulating interactions in natural microecosystems. Traditional single technologies fail to achieve precise design and regulation of synthetic biological systems. However, microbiomics, which integrates multi-omics, spatial omics, and culturomics, provides a full-chain solution for synthetic biology covering “functional prediction-dynamic regulation-functional verification-community construction” by decoding multidimensional molecular information, spatial distribution patterns, and cultivation interaction mechanisms of biological systems. This chapter systematically elaborates on how integrated multi-omics guides the optimization of metabolic pathways in synthetic biological systems, how spatial omics deciphers the spatial interaction mechanisms of synthetic communities, and how innovations in culturomics break through the cultivation bottlenecks of synthetic strains. Ultimately, it realizes the in-depth integration of omics technologies and synthetic biology, thereby promoting the industrial application of synthetic biological systems in fields such as biomanufacturing, environmental remediation, and medical health.