This paper describes a novel microfluidic cell culture platform for 3-dimensional cell patterning and in situ cell-based assays. The microfluidic device is fabricated by photolithography and poly(dimethylsiloxane) (PDMS) replica molding. The mixture of HepG2 cells and a sol-gel transition peptide hydrogel is hydrodynamically focused and patterned in the middle of the main channel of a microfluidic device. The cells are 3-dimensionally encapsulated and cultured on a microscope plate for over 2 days at the same time. A toxicant injected into one side inlet of a microfluidic device shows a linear concentration gradient profile at the entire area in the stripe-shaped micro-scaffold. The higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and provides credible cell-based screening and assays including cytotoxicity test, cell viability monitoring, continuous dose-response assay, drug-drug interaction and metabolism analysis. The device is also contributed to a novel biomimetic blood vessel platform for micro-angiological study by exchanging the mixture and media flows. Various geometries of vessel walls and blood flow in a biomimetic blood vessel model can be controlled by the media, distilled water and media flows in a microfluidic device.