To fabricate the μPAD, the wax dipping method was employed to combine a blood separation membrane with patterned Whatman No.1 paper. The LF1 membrane was found to be the most suitable for blood separation on the μPAD when fabricated by wax dipping. With this membrane, blood cells (both red and white) were trapped on the blood separation membrane, allowing pure plasma to flow to the detection zone through capillary force. The LF1-μPAD was found to be functional with human whole blood of 24-55% hematocrit without the need for dilution. Microscopy confirmed that the device isolated plasma with high purity, without blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was found to be 2.62% (n = 10) and 5.84% (n = 30) for within-day and between-day precision, respectively. The proposed blood separation on the μPAD has the potential to reduce turnaround time, sample volume, sample preparation, and detection processes for clinical diagnosis and point-of-care testing. By combining a blood separation membrane with a filter membrane and using the wax dipping method, the chemistry surface of the paper is conserved, and the capillary force action of plasma occurs without the need for external pumping force. Moreover, the passive capillary force of the device eliminates the blood cell damage problems that occur with pressure-driven flow.