A portable self-flow filtration and separation platform was designed using soft lithography to create a polydimethylsiloxane (PDMS) microfluidic channel cover combined with a matching acrylic substrate. The separation zone was filled with microbeads of appropriate sizes to achieve universal filtration and separation. This simple structure requires only 20 μl of the sample for filtration separation. A vacuum of 760 torr is applied to the porous PDMS cover to drive the sample during testing. The average time required for a 20 μl sample of blood to pass through the separation zone is about 56 s, while the filling time for the detection zone of volume 6 μl is about 319 s. When the hematocrit of the blood sample is about 20-25%, the separation efficiency is 99.98%. Further, the separation efficiency of fat globules from raw milk is close to 100%, whereas almost all impurities are filtered out from juice and stool samples. It is also observed that E. coli in the stool can pass from the separation to detection zone at a maximum rate of about 81.21%, with an average of about 68.18%.