We report the use of a laser-based direct-write (LDW) technique that allows the design and fabrication of three-dimensional (3D) structures within a paper substrate that enables implementation of multi-step analytical assays via a 3D protocol. The technique is based on laser-induced photo-polymerisation, and through adjustment of the laser writing parameters such as the laser power and scan speed we can control the depths of hydrophobic barriers that are formed within a substrate which, when carefully designed and integrated, produce 3D flow paths. So far, we have successfully used this depth-variable patterning protocol for stacking and sealing of multi-layer substrates, for assembly of backing layers for two-dimensional (2D) lateral flow devices and finally for fabrication of 3D devices. Since the 3D flow paths can also be formed via a single laser-writing process by controlling the patterning parameters, this is a distinct improvement over other methods that require multiple complicated and repetitive assembly procedures. This technique is therefore suitable for cheap, rapid and large-scale fabrication of 3D paper-based microfluidic devices.