In recent years, the study of complex cellular interactions has been hampered by the limitations of traditional two-dimensional (2D) and single-cell type culture systems, which fail to accurately mimic the intricate dynamics of human tissues. To bridge this gap, assembloid technology has emerged as a transformative approach. Assembloids are self-organizing three-dimensional (3D) systems formed by integrating multiple organoids or cell types, providing a more accurate model for studying inter-tissue and inter-organ communication. Here, we categorize current assembloids into four types based on assembly strategies—multi-region, multi-lineage, multi-gradient, and multi-layer—each designed to replicate specific biological phenomena with high fidelity. We also explore the diverse applications of assembloids across various human systems, demonstrating the broad application scope of assembloids. Finally, we highlight the challenges faced by assembloid technology and outline its future prospects. Overall, assembloids represent a powerful platform for advancing research in developmental biology, disease modeling, and drug discovery.