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 a pressure of 3.8 MPa, temperature of 80◦C, and 3 min 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. The wicking speed in the fabricated paper strips was slightly lower than that of non-processed paper, resulting from a reduced paper pore size after hot pressing. 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 a two-dimensional paper-based device and flowing in a three-dimensional counterpart were thoroughly investigated, demonstrating that the paper devices from this fabrication process are potentially applicable as analytical devices for biomolecule detection. Fast, easy, and inexpensive parafilm hot press fabrication presents an opportunity for researchers to develop paperbased analytical devices in resource-limited environments.