Sample preparation steps (e.g., preconcentration and separation) are key to enhancing sensitivity and reliability in biomedical and analytical chemistry. However, conventional methods (e.g., ultracentrifugation) cause significant loss of sample as well as their contamination. In this study, we developed a paper-based three-dimensional (3D) origami ion concentration polarization preconcentrator (POP) for highly efficient and facile sample preparation. The unique design of POP enables simultaneous preconcentration and spatial separation of target analytes rapidly and economically. The POP comprises accordion-like multifolded layers with convergent wicking areas that can separate analytes based on their sizes in different layers, which can then be easily isolated by unfolding the POP. We first demonstrated 100-fold preconcentration of albumin and its isolation on the specific layers. Then, we demonstrated the simultaneous preconcentration and spatial separation of microspheres of three different sizes (with diameters of 0.02, 0.2, and 2 μm) on the different layers.