We developed a simple method to achieve semiquantitative detection of an amphiphilic biosample through measuring the length of flow on a microfluidic analytical device (μPAD) based on paper. When an amphiphilic sample was dripped into a straight microchannel defined with a printed wax barrier (hydrophobic) on filter paper (hydrophilic), the length of flow was affected by the reciprocal effect between the sample, the filter-paper channel and the wax barrier. The flow length decreased with increasing concentration of an amphiphilic sample because of adsorption of the sample on the hydrophobic barrier. Measurement of the flow length enabled a determination of the concentration of the amphiphilic sample. The several tested samples included surfactants (Tween 20 and Triton X-100), oligonucleotides (DNA), bovine serum albumin (BSA), human albumin, nitrite, glucose and low-density lipoprotein (LDL). The results show that the measurement of the flow length determined directly the concentration of an amphiphilic sample, whereas a non-amphiphilic sample was not amenable to this method. The proposed method features the advantages of small cost, simplicity, convenience, directness, rapidity (<5 min) and requirement of only a small volume (5 μL) of sample, with prospective applications in developing areas and sites near patients for testing at a point of care (POCT).