This article describes a new method, referred to as "tear-off patterning," for patterning nitrocellulose (NC) membranes in order to fabricate NC-based point-of-care (POC) diagnostic devices. Paper-based microfluidic sensors usually employ hydrophobic barrier coatings such as paraffin wax on either paper or membranes. Herein, complex patterns were fabricated by stamping the target area with dimethyl sulfoxide before tearing off the stamped area. Fluid flow and morphological analyses were performed in order to characterize the patterned membranes. Furthermore, the myoglobin and creatine kinase-MB levels in human serum were measured simultaneously using a dual-fluidic-channel-patterned NC membrane in order to confirm the usefulness of the patterning method for fabricating POC biosensors. The proposed method for patterning NC membranes offers clear advantages, such as the ability to fabricate complex designs and patterns without a hydrophobic barrier after protein immobilization in a laboratory and in a simple, low-cost manner. We believe that this method can be used to develop various POC diagnostic biosensors at the research and development stage and can help improve the performance and features of POC diagnostic devices.