Diagnosis sensors using micro-total analysis systems (m-TAS) have been developed for detecting target biomolecules such as proteins and saccharides because they are signal biomolecules for monitoring body conditions and diseases. Valve systems that regulate the flow of a sample solution in the microchannels are the most important elements in fabricating m-TAS as diagnostic chips. Standard m-TAS uses electromechanical elements fabricated by MEMS technologies. As such electromechanical elements are based on complicated systems, advantages of m-TAS such as simplicity and miniaturization are lost. We have prepared biomolecularly stimuli-responsive hydrogels using biomolecular complexes as dynamic crosslinks that reversibly form or dissociate according to changes in an external environment. For example, biomolecularly stimuli-responsive hydrogels with antigen-antibody complexes or glycopolymer-lectin complexes swelled gradually in response to the concentration of a target biomolecule such as an antigen and glucose. Biomolecularly stimuli-responsive hydrogels that exhibited unique shrinkage in the presence of a target biomolecule were strategically designed by molecular imprinting. Their biomolecularly stimuli-responsive swelling and shrinkage were based on drastic changes in the crosslinking density of the hydrogel networks, induced by the dissociation and formation of biomolecular complexes as dynamic crosslinks in the presence of a target biomolecule. In this study, three types of micron-sized molecular stimuli-responsive hydrogels, bisphenol A (BPA)-, glucose- and concanavalin A (ConA)-responsive micro-hydrogels, were prepared by photopolymerization in a microchannel. We investigated the flow rate of the microchannels with their responsive micro-hydrogels in the absence and presence of the target molecule. ConA-responsive micro-hydrogels were prepared by molecular imprinting as follows. First, glycosyloxyethyl methacrylate as a ligand monomer and ConA as a molecular template were mixed for forming a molecular complex. Acrylamide as a main monomer, N, N’-methylenbisacrylamide as a crosslinker, and 2, 2'-azobis(2-methylpropionamidine) dihydrochloride as a photoinitiator were dissolved in a phosphate buffer solution. The solution was exposed to UV light focused using an inverted fluorescence microscope to polymerize the monomers. A ConA-imprinted micro-hydrogel was prepared near the branch point of the Y-shaped microchannel. To evaluated the ConA-imprinted micro-hydrogel as a self-regulated valve for a microchannel flow control system, the flow rates of the channels were measured while a buffer solution without and with target ConA was pumped into the Y-shape microchannel. After flowing a buffer solution with ConA, the ConA-imprinted micro-hydrogel in the channel shrank in response to ConA, followed by an increase in the flow rate. These results suggest that the ConA-imprinted micro-hydrogel is useful as a self-regulated micro-hydrogel valve for fabricating smart microchannels which can autonomously change the flow rate in response to the target ConA. ConA-responsive micro-hydrogels leads to the fabrication of smart microchannels in which micro-hydrogel valves can open and close autonomously and sensitively. In addition, BPA-responsive and glucose-responsive micro-hyrogels were prepared in a microchannel by photopolymerization using an inverted fluorescence microscope. This paper also demonstrates the autonomous changes in the flow rate in the microchannel with their micro-hydrogels as molecularly stimuli-responsive valves.