Since the monumental work conducted by Whitesides et al. in 2007, research and development of paper-based microfluidics has been widely carried out, with its applications ranging from chemical and biological detection and analysis, to environmental monitoring and food-safety inspection. Paper-based microfluidics possesses several competitive advantages over other substrate materials, such as being simple, inexpensive, power-free for fluid transport, lightweight, biodegradable, biocompatible, good for colorimetric tests, flammable for easy disposal of used paper-based diagnostic devices by incineration, and being chemically modifiable. Myriad methods have been demonstrated to fabricate paper-based microfluidics, such as solid wax printing, cutting, photolithography, microembossing, etc. In this study, fabrication of paper-based microfluidics was demonstrated by spray on the printed paper. Different from the normally used filter papers, printing paper, which is much more accessible and cheaper, was utilized as the substrate material. The toner was intended to serve as the mask and the patterned hydrophobic barrier was formed after spray and heating. The processing parameters such as toner coverage on the printing paper, properties of the hydrophobic spray, surface properties of the paper, and curing temperature and time were systematically investigated. It was found that, after repetitive printing four times, the toner was able to prevent the hydrophobic spray (the mixture of PDMS and ethyl acetate) from wicking through the printing paper. The overall processing time for fabrication of paper-based microfluidic chips was less than 10 min and the technique is potentially scalable. Glucose detection was conducted using the microfluidic paper-based analytical devices (µPADs) as fabricated and a linear relationship was obtained between 1 and 10 mM.