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The use of microfluidic systems is a recent trend in diagnostic assays and bioreactors based on protein and cellular engineering. Most microfluidic biodevices would require combination with a technique to pattern proteins and living cells within microchannels just prior to the use of the devices, since these bio-elements are delicate against desiccation, oxidation and heat. Here, we present a simple, electrochemical method to locally immobilize proteins and cells on substrate surface, easily applicable to the microfluidic system. The integration of the technique into a microfluidic device and the on-demand immobilization of proteins and cells were achieved. The sandwich immunoassay within the microchannel was demonstrated, in which a series of protocols from immobilization to measurement can be carried out under physiochemical conditions.