The concentrations of nitrite and thiocyanate in saliva can be used as the biomarkers of the progression of periodontitis disease and environmental tobacco smoke exposure, respectively. Therefore, it is particularly necessary to detect these two indicators in saliva. Herein, the three-dimensional single-layered paper-based microfluidic analytical devices (3D sl-μPADs) were, for the first time, fabricated by the spraying technique for the colorimetric detection of nitrite and thiocyanate at the same time. The conditions for 3D sl-μPADs fabrication were optimized in order to well control the penetration depth of the lacquer in a paper substrate. Then, the developed 3D sl-μPADs were utilized to simultaneously detect nitrite and thiocyanate and the limits of detection are 0.0096 and 0.074 mM, respectively. What is more, the μPADs exhibited good specificity, good repeatability, and acceptable recoveries in artificial saliva. Therefore, the developed 3D sl-μPADs show a great potential to determine nitrite and thiocyanate for the assessment of the human health.