This paper describes a portable, three-dimensional (3D) microfluidic paper-based analytical device (3D-μPAD) for performing enzyme-linked immunosorbent assays (ELISA). To run an ELISA, the device requires only the addition of a small drop (2 μL per test zone) of sample and six drops (75 μL in total volume) of buffer solution. All the reagents required for the analysis are stored in dry form within the device, and are dissolved in the buffer and delivered to the test zones during the assay. The innovative feature of the device, which enables the delivery of different reagents and washes to the test zones without cross contamination, is a movable strip containing the test zones that can be moved manually through the device, and stops only at specified points where the test zones come into contact with different fluidic paths that sequentially transfer (i.e., wash) the reagents into the test zones. Using rabbit IgG as a model analyte, we performed an ELISA in 43 minutes, with a detection limit of 330 pM. We also demonstrated the detection of hepatitis B surface antigen (HBsAg) in serum.