Cell–cell interaction accounts for one of the most influential factors affecting the viability and functionality of cell‐based tissue models. In this respect, various methods capable of producing micro‐patterns with cell spheroids are introduced to simultaneously improve contact‐dependent and ‐independent cell‐cell interactions. However, no method has yet been designed to effectively generate precise 3D patterns with multiple spheroid types. In this study, a new high‐precision and convenient 3D spheroid printing technology is developed, designated as 3D bio‐dot printing. This new technique is designed to produce cell‐laden, non‐adhesive micro‐pores within 3D structures to allow cell spheroids to be induced at printed sites. Experimental results show that various cell types, including hepatocytes, pancreatic β‐cells, and breast cancer cells, can be employed for the in situ formation of cell spheroids, and 3D freeform structures with multiple spheroid types can be printed. Moreover, this novel technology can also be used for performing 3D invasion assays. More importantly, it ensures that the precise control of spheroid size and position is achieved at micrometer scale. Finally, the usefulness of this novel technology is demonstrated by producing multicellular micro‐patterns with primary hepatocyte spheroids and endothelial cells, that exhibit significantly improved long‐term hepatic function and drug metabolism. A new, high‐precision 3D cell spheroid printing technique, named 3D bio‐dot printing, is presented. The new technique exhibits convenient, precise, and 3D producible results with multiple cell spheroids, and can be applied to 3D spheroid invasion assays. Its application to hepatic models demonstrates that the technique can significantly improve long‐term hepatic function and drug metabolism through precise patterning with cell spheroids.