The micro-patterned poly(ethylene glycol) (PEG) brush surface for controlling protein and cell interactions was constructed. PEGylated substrates with two-dimensional arrays of plasma-etched circular domains were prepared by coating of mercapto-functionalized (PEG) on Au surface, followed by plasma-etching through a metal mask pattern with circular holes. The micro-patterning of bovine articular chondrocytes or rat primary hepatocytes hetero-spheroids underlaid with human umbilical endothelial cells (HUVEC) was achieved. These arrayed spheroids are promising materials for tissue and cell-based biosensors (TBB and CBB) as well as tissue engineering technologies.