As an important target of point-of-care diagnostics, hematocrit is closely related to the health condition of human beings. Here, we developed a paper microfluidic chip for quantitative hematocrit measurement. The paper microfluidic chips were pretreated by sodium chloride, which would cause the dehydration of red blood cells and affect their flow abilities in the porous nitrocellulose paper. Plasma flowed ahead of red blood cells and separated from whole blood sample. We used the area of plasma region as the index to refer to the hematocrit, and built a linear calibration curve of plasma area versus hematocrit with R 2 > 0.92 for blood samples with hematocrit ranging from 30% to 60%, which covers the clinically relevant hematocrit range.