Compared to 2D monolayer of cells, arranged 3D cell spheroids represents higher tissue specific function. Not only the form of cell spheroids, but spatial arrangement with multiple cell types has been emerged for in vivo relevance. However, conventional techniques to arrange 3D spheroids in hydrogel with multiple cells had challenges: (1) Spheroid production and 3D fabrication steps were conducted individually. (2) Methods for patterning spheroids with other cells required multiple microfabrication processes. Here, a novel bioprinting process has been developed that can simultaneously perform 3D micro-positioning and formation of spheroids. Poly(ε-caprolactone) (PCL) was firstly printed to support hydrogel structurally. After that, matrix bio-ink was printed followed by dispensing cell laden sacrificial bio-ink spherically into the matrix hydrogel. Un-crosslinked materials in matrix hydrogel were removed in an incubator, which provided a space for in situ cell spheroid formation. By modulating printing parameters, size of cell spheroids was controlled. A path generation software enabled spheroids positioned in controlled interval. 3D constructions containing spheroids patterned showed great cytocompatibility and functionality. Lastly, hepatocyte spheroids and endothelial cells were arranged spatially, which improved hepatic functionality. Our new method would be used as a powerful tool in development of artificial tissue and model for drug screening.