We present novel redox-responsive hydrogels based on poly(N-isopropylacrylamide) or poly(acrylamide), consisting of a reversible disulfide crosslinking agent N,N '-bis(acryloyl)cystamine and a permanent crosslinking agent N,N '-methylenebisacrylamide for microfluidic applications. The mechanism of swelling/deswelling behavior starts with the cleavage and reformation of disulfide bonds, leading to a change of crosslinking density and crosslinking points. Raman and ultraviolet-visible spectroscopy confirm that conversion efficiency of thiol-disulfide interchange up to 99%. Rheological analysis reveals that the E modulus of hydrogel is dependent on the crosslinking density and can be repeatedly manipulated between high- and low-stiffness states over at least 5 cycles without significant decrease. Kinetic studies showed that the mechanical strength of the gels changes as the redox reaction proceeds. This process is much faster than the autonomous diffusion in the hydrogel. Moreover, cooperative diffusion coefficient (D-coop) indicates that the swelling process of the hydrogel is affected by the reduction reaction. Finally, this reversibly switchable redox behavior of bulky hydrogel could be proven in microstructured hydrogel dots through short-term photopatterning process. These hydrogel dots on glass substrates also showed the desired short response time on cyclic swelling and shrinking processes known from downsized hydrogel shapes. Such stimuli-responsive hydrogels with redox-sensitive crosslinkers open a new pathway in exchanging analytes for sensing and separating in microfluidics applications. (c) 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019