Multicellular structures are organisms consisting of cell populations, and constructing multicellular structure is of great importance for the development of tissue engineering and regenerative medicine. The use of self-organization processes in three-dimensional (3D) culture is receiving increased attention for bio-manufacturing and regenerating injured tissue. This review covers the research work on the fundamental and key techniques regarding 3D cellular self-assembly. It includes the 3D culture of cells inside bio- compatible hydrogels, controllable formation of 3D multicellular structure, and the associated Turing reaction-diffusion mechanism. In order to further study the mechanism of 3D self-assembly of cells and find a reliable way to regulate this self-organization in 3D extracellular matrix with spatial gradients of the concentrations of exogenous and cell-secreted factors, this review at last proposes to study the 3D models of cellular self-assembling inside structurally graded hydrogel, so that the development of rational regulation technology for 3D multicellular structure and tissue bud could be facilitated.