Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these applications it has become increasingly apparent that the spatiotemporal arrangement of biological molecules in vitro is important for the investigation of the cellular functions found in vivo. However, the cell patterning techniques often used are limited to creating 2D functional surfaces on glass and silicon. In addition, in general, these procedures are not easy to implement in conventional biological laboratories. Here, we show the formation of a living poly(ethylene glycol) (PEG) layer that can be patterned with visible light on plastic surfaces. This new and simple method can be expanded to pattern multiple types of biomolecule on either a previously formed PEG layer or a plastic substrate. Using common plastic wares (i.e., polyethylene films and polystyrene cell culture Petri-dishes), we demonstrate that these PEG-modified surfaces have a high resistance to protein adsorption and cell adhesion, while at the same time, being capable of undergoing further molecular grafting with bioactive motifs. With a photomask and a fluid delivery system, we illustrate a flexible way to immobilize biological functions with a high degree of 2D and 3D spatial control. We anticipate that our method can be easily implemented in a typical life science laboratory (without the need for specialized lithography equipment) offering the prospect of imparting desirable properties to plastic products, for example, the creation of functional microenvironments in biological studies or reducing biological adhesion to surfaces.