In this work, we report a one-step approach for fabricating screened-printed microfluidic paper-based analytical devices (μPADs) using polylactic acid as a new hydrophobic material. A polylactic acid solution was screen printed onto chromatography papers to create hydrophobic patterns for fluidic channels. The optimal polylactic acid concentration for successful device fabrication is 9% w/v. The μPADs were fabricated within 2 min and provided high reproducibility and stability. The utility of polylactic acid screen-printing was demonstrated for the simultaneous detection of nitrite and nitrate using colorimetric detection. Under optimized experimental conditions, the detection limits and the linear ranges, respectively, were 1.2 mg L⁻¹ and 2–10 mg L⁻¹ for nitrite and 3.6 mg L⁻¹ and 10–50 mg L⁻¹ for nitrate. The detection times for both ions were found to be within 12 min. The developed μPAD was applied for the simultaneous determination of these ions in food samples, and no significant differences in the analytical results were observed compared to those of the reference method. The polylactic acid screen-printing approach presented here provides a simple, rapid, and cost-effective alternative fabrication method for fabricating μPADs.