Paper-based analytical devices have been substantially developed in recent decades. Many fabrication techniques for paper-based analytical devices have been demonstrated and reported. Herein we report a relatively rapid, simple, and inexpensive method for fabricating paper-based analytical devices using parafilm hot pressing. We studied and optimized the effect of the key fabrication parameters, namely pressure, temperature, and pressing time. We discerned the optimal conditions, including pressure of 3.8 MPa (3 tons), temperature of 80oC, and 3 minutes of pressing time, with the smallest hydrophobic barrier size (821 µm) being governed by laminate mask and parafilm dispersal from pressure and heat. Physical and biochemical properties were evaluated to substantiate the paper functionality for analytical devices. Wicking speed in the fabricated paper strips was slightly slower than that of non-processed paper, resulting from reducing paper pore size. A colorimetric immunological assay was performed to demonstrate the protein binding capacity of the paper-based device after exposure to pressure and heat from the fabrication. Moreover, mixing in two-dimensional paper-based device and flowing in a three-dimensional counterpart were thoroughly investigated, demonstrating that the paper device from this fabrication process is potentially applicable as analytical devices for biomolecule detection. Fast, easy, and inexpensive parafilm hot press fabrication presents an opportunity for researchers to develop paper-based analytical devices in resource-limited environments.