Cell-free (cf) nucleic acids are considered important and have been used as selective biomarkers. Conventional techniques for cf nucleic acid biomarker isolation from blood are generally time-consuming, complicated, and expensive. This study describes a lab-on-a-disk system equipped with newly developed immiscible filtration assisted by surface tension (IFAST), which can achieve the rapid isolation of cfDNA from whole blood. The principle of centrifugal IFAST (C-IFAST) is introduced. An arch-like channel for magnetic bead transfer in the immiscible phase is designed, which builds both a virtual water–air “wall” and an air–oil “wall” to prevent the blending of water and oil. The entire process requires less than 15 min and achieves the recovery of 65% of cfDNA from plasma and 30% from whole blood. Experiments were performed to test the validity of the chip, showing that this technique takes less time to obtain results of identical quality compared to commercial kits. The proposed C-IFAST method enables rapid and reliable cfDNA isolation from large whole blood volume (4 ml) and can potentially be used in “liquid biopsy” point-of-care diagnosis.