In this article, we describe a facile method to fabricate MIP patterns in specifically designed microfluidic channels. With this design, homogenous and stable MIP films were spatially immobilized inside the patterned PDMS channels. In this system, the fluorescent response was identified by detection of the fluorescence-labeled bovine serum albumin (BSA) template. In comparison, non-imprinted polymer (NIP) was also prepared. From the results of fluorescent response, significant binding behaviors of BSA molecules into the cavities of MIP patterns was observed due to the increased residence time in each ancillary hexagonal channel caused by the turbulent flow. However, the NIP patterns did not show the fluorescent response. Thus, the use of this system provides effective MIP-based microfluidic channels for the application of biosensors.