This paper describes the development of a novel paper-based capillary electrophoresis (pCE) microdevice using mineral paper, which is durable, oil and tear resistant, and waterproof. The pCE device is inexpensive (similar to$1.6 per device for materials), simple to fabricate, lightweight, and disposable, so it is more adequate for point-of care (POC) pathogen diagnostics than a conventional CE device made of glass, quartz, silicon or polymer. In addition, the entire fabrication process can be completed within 1 h without using expensive clean room facilities and cumbersome photolithography procedures. A simple cross-designed pCE device was patterned on the mineral paper by using a plotter, and assembled with an OHP film via a double-sided adhesive film. After filling the microchannel with polyacrylamide gel, the injection, backbiasirig, and separation steps were sequentially operated to differentiate single-stranded DNA (ssDNA) with 4 bp resolution in a 2.9 cm-long CE separation channel. Furthermore, we successfully demonstrated the identification of the PCR amplicons of two target genes of Escherichia coli O157:H7 (rrsH gene, 121 bp) and Staphylococcus aureus (glnA gene, 225 bp). For accurate assignment of the peaks in the electropherogram, two bracket ladders (80 bp for the shortest and 326 bp for the longest) were employed, so the two amplicons of the pathogens were precisely identified on a pCE chip within 3 min using the relative migration time ratio without effect of the CE environments. Thus, we believe that the pCE microdevice could be very useful for the separation of nucleic acids, amino acids, and ions as an analytical tool for use in the medical applications in the resource-limited environments as well as fundamental research fields.