To transform from reactive to proactive healthcare, there is an increasing need for low-cost and portable assays to continuously perform health measurements. The paper-based analytical devices could be a potential fit for this need. To miniaturize the multiplex paper-based microfluidic analytical devices and minimize reagent use, a fabrication method with high resolution along with low fabrication cost should be developed. Here, we present an approach that uses a desktop pen plotter and a high-resolution technical pen for plotting high-resolution patterns to fabricate miniaturized paper-based microfluidic devices with hundreds of detection zones to conduct different assays. In order to create a functional multiplex paper-based analytical device, the hydrophobic solution is patterned on the cellulose paper and the reagents are deposited in the patterned detection zones using the technical pens. We demonstrated the effect of paper substrate thickness on the resolution of patterns by investigating the resolution of patterns on a chromatography paper with altered effective thickness. As the characteristics of the cellulose paper substrate such as thickness, resolution, and homogeneity of pore structure affect the obtained patterning resolution, we used regenerated cellulose paper to fabricate detection zones with a diameter as small as 0.8 mm. Moreover, in order to fabricate a miniaturized multiplex paper-based device, we optimized packing of the detection zones. We also showed the capability of the presented method for fabrication of 3D paper-based microfluidic devices with hundreds of detection zones for conducting colorimetric assays.