This study demonstrates a simple approach for fabricating a 3D-μPAD from a single sheet of paper by double-sided printing and lamination. First, a wax printer prints vertically symmetrical and asymmetrical wax patterns onto a double-sided paper surface. Then, a laminator melts the printed wax patterns to form microfluidic channels in the paper sheet. The vertically symmetrical wax patterns form vertical channels when the melted wax patterns make contact with each other. The asymmetrical wax patterns form lateral and vertical channels at the cross section of the paper when the printed wax patterns are melted to a lower height than the thickness of the single sheet of paper. Finally, the two types of wax patterns form a 3D microfluidic network to move fluid laterally and vertically in the single sheet of paper. This method eliminates major technical hurdles related to the complicated and tedious alignment, assembly, bonding, and punching process. This 3D-μPAD can be used in a multiplex digital assay to measure the concentration of a target analyte in a sample solution simply by counting the number of colored bars at a fixed time. It does not require any external instruments to perform digital measurements. Therefore, we expect that this approach could be an instrument-free assay format for use in developing countries.