Extracellular vesicles (EVs) play a significant role in the pathophysiological process of many diseases, highlighting their values in medical diagnosis and disease monitoring. However, the current EV isolation methods are time-consuming, inconvenient to operate, and incompatible with downstream analyses. Here, we present a novel isolation method employing anionic polysaccharide-modified filter papers for the isolation of EVs (AppiEV) via electrostatic adsorption. A disc of glass fiber-based filter modified with sodium alginate was assembled into a spin column to function as the solid capture phase. In the acidic condition, EVs were induced to carry more positively charged ions, which enable the capture of EVs by the negatively charged filter paper. After a wash, the EVs were released from the spin column using an alkaline elution buffer, which induces the EVs to carry more negative charges. The EVs isolated by AppiEV from cell culture supernatants, plasma, and urine are similar to or even better than those isolated by ultracentrifugation in terms of EV size distribution, protein distribution, and nucleic acid contents. Due to the interference removal of the EV-free RNA and DNA attributed to the negatively charged capture medium, the eluate of AppiEV could be directly used for genetic analysis, including the stem-loop RT-PCR analysis of miR-21 and the allele-specific PCR analysis of mutation genes of EGFR p.L858R and EGFR p.T790M. We believe that AppiEV offers a simple and efficient approach for the isolation of high-quality EVs from various liquid specimens.