In this paper, we propose a new fabrication method of pH-responsive microfluidic valves and suggest the feasibility as a microminiature systems without peripheral devices. Conventionally, in-situ photopolymerization method has been employed to build the micro functional structure inside microfluidic channel. Here, we suggest a new method allowing the mass production of stimuli-responsive microsystem through the use of pre-made microstructure as actuating element like assembly of commercial products in the factory. We have massively fabricated pH-responsive microspheres that act as actuating component through the use of PDMSbased microfluidic apparatus and ‘on the fly’ photopolymerization method. By the incorporation of this microsphere into the microvalve during the fabrication process, we have produced the pH-responsive microfluidic valve in a simple way. The operation and the function of the fabricated microvalve were evaluated through the diverse experiments. The microvalve was successfully fabricated, and functioned well according to the pH change.