A portable microplasma generation device (MGD) operated in ambient air is introduced for making a microfluidic paper-based analytical device (μPAD) that serves as a primary healthcare platform. By utilizing a printed circuit board fabrication process, a flexible and lightweight MGD can be fabricated within 30 min with ultra low-cost. This MGD can be driven by a portable power supply (less than two pounds), which can be powered using 12 V-batteries or ac-dc converters. This MGD is used to perform maskless patterning of hydrophilic patterns with sub-millimeter spatial resolution on hydrophobic paper substrates with good pattern transfer fidelity. Using this MGD to fabricate μPADs is demonstrated. With a proper design of the MGD electrode geometry, μPADs with 500-μm-wide flow channels can be fabricated within 1 min and with a cost of less than $USD 0.05/device. We then test the μPADs by performing quantitative colorimetric assay tests and establish a calibration curve for detection of glucose and nitrite. The results show a linear response to a glucose assay for 1-50 mM and a nitrite assay for 0.1-5 mM. The low cost, miniaturized, and portable MGD can be used to fabricate μPADs on demand, which is suitable for in-field diagnostic tests. We believe this concept brings impact to the field of biomedical analysis, environmental monitoring, and food safety survey.