Microvesicles and exosomes are promising liquid biopsy biomarkers. However, conventional isolation techniques damage and contaminate the biomarkers. We developed an origami-paper-based device for effective isolation of biomarkers with less damage and in fewer steps. The multi-folded device enables the preconcentration of the microvesicles/exosomes on specific layers (∼5-fold) by the ion concentration polarization technique and they were simply isolated from the rest of the sample by unfolding the device.