Micropatterned PEGylated substrates with two-dimensional arrays of plasma-etched circular domains (diameter:100 micro-m) were prepared by coating of mercapto-functionalized poly(ethylene glycol) (PEG) on Au surface, followed by plasma-etching through a metal mask pattern with circular holes. The PEGylated region on the patterned substrate works to repel proteins, consequently, inhibits cell adhesion. Then the micro-patterning of bovine articular chondrocytes or rat primary hepatocytes hetero-spheroids underlaid with human umbilical endothelial cells (HUVEC) was achieved on the plasma-etched circular domains, exposing the base gold surface. Obtained results suggested that the efficiency of inhibiting non-specific protein adsorption significantly affects on construction of micro-patterned cell adhesion and hetero-spheroids. The formation of hetero-spheroid thus suggested is significantly modulated by suface properties, particularly non-fouling character of PEG region. These arrayed spheroids is promising materials for tissue and cell-based biosensors (TBB/CBB) as well as tissue engineering technologies.