Small extracellular vesicles (sEVs) play important roles in mediating intercellular communication and regulating biological processes. Facile sEV isolation is the essential and preliminary issue for their function investigation and downstream biomedical applications, while the traditional methods are challenged by tedious procedures, low purity, low yield, and potential damage. In this work, we developed an sEV isolation paper-based device (sEV-IsoPD) based on a three-dimensional (3D) paper chip, which is composed of a porous membrane for size exclusion and a metal-organic framework (MOF)/antibody-modified paper for immunoaffinity capture. In combination with a peristaltic pump-driven flow system, the sEV-IsoPD can efficiently isolate EV from cell culture medium and serum. Compared with the ultracentrifugation method, sEV-IsoPD exhibited a 5.1 times higher yield (1.7 × 109 mL-1), 1.6 times higher purity (1.6 × 1011 mg-1), and 7.5 times higher recovery (77.3%) with only 8.3% of the time (30 min) and 1.0% of the instrument cost ($710). Moreover, sEV concentration can be visually detected in a quantitative manner with this paper-based device with a linear range from 3.0 × 106 to 3.0 × 1010 mL-1 and a detection limit of 2.2 × 106 mL-1. The sEV-IsoPD provides an efficient and practical approach for the rapid isolation and visible detection of sEVs, which are promising for the preparation of sEVs and diagnosis of disease.