This paper presents a device (“fluidic biomill”) and method for integrated mechanical structuring and biofunctionalization of microfluidic channels, enabling rapid prototyping of biochips. The fluidic biomill leverages hydrodynamic flow confinements (HFCs) to localize the interaction between a fluid and a substrate to a microscale region. By confining organic solvents, and scanning with the biomill over the substrate surface, microchannels as deep as 90 µm are fluidically milled. Subsequently, by switching the confined fluid from a milling solution to a patterning solution, biomolecules are immobilized on the surface of the microchannel. An in-depth analysis of fluidic milling rates and milled microchannel properties is presented, along with a discussion on the dominant factors in controlling the dimensions of the fluidically milled channel. To demonstrate the utility of this method, a biotin functionalized biochip is fabricated in less than 5 min out of a polystyrene slide and its functionalities are validated by implementing on-chip electrokinetic sample focusing, pressure driven flow, and a surface-based reaction.