A two-dimensional microarray of ten thousand (100×100) hepatocyte hetero-spheroids, underlaid with non-parenchymal cells, was successfully constructed with a 100-µm spacing on micro-fabricated glass substrates that were coated with poly(ethylene glycol) (PEG). Co-cultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100-µm, functioning as a miniaturized liver to secrete albumin for at least 1 month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors (TBB and CBB), detecting a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.