One of the primary challenges for the development of electrochemical sensors for dopamine (DA) is the lack of selectivity due to the major interference from ascorbic acid (AA) which is oxidized at a potential close to the working potential of DA. In this work, we present a means for simple and cost-effective electrochemical detection of DA in the presence of interference using a microfluidic paper-based analytical device (eμPAD) to improve the selectivity and sensitivity. The device was made by incorporating the sample separation and the electrochemical detection elements into a single device, which required a single drop of the sample solution. For sample separation, Nafion was cast on the sample zone and microfluidic channel to block AA, before the solution was allowed to continuously flow to the detection zone. A multilayered eμPAD was fabricated by placing a round cotton pad on the detection zone to enable the generation of a quasi-steady flow rate. Thereafter, a packing tape was used to seal the top part of the device to increase the flow rate and prevent volatilization of the sample on the eμPAD. The concentration of DA that could be measured by the poly(glutamic acid) modified screen-printed graphene electrode was in the range of 1–200 μM, and the limit of detection (LOD) was 0.41 μM (3SD/slope). The proposed platform provides an accurate and satisfactory method for the detection of DA in real samples compared with UV-Vis spectroscopy.