A lateral flow assay using DNA aptamer-based sensing for the detection of dopamine in urine is reported. Dopamine duplex aptamers (hybridized sensor with capture probe) are conjugated to 40-nm Au nanoparticles (AuNPs) with 20T linkers. The detection method is based on the dissociation of the duplex aptamer in the presence of dopamine, with the sensor part undergoing conformational changes and being released from the capture part. Hybridization between the complementary DNA in the test line and the conjugated AuNP-capture DNA produces a red band, whose intensity is related to the dopamine concentration. The minimum detectable concentration obtained by ImageJ analysis was <10 ng/mL (65.2 nM), while the visual limit of detection is estimated to be ~50 ng/mL (normal range of dopamine in urine of 52-480 ng/mL or 0.3-3.13 μM). No cross reactivity to other stress biomarkers in urine was confirmed. These results indicate that this robust and user-friendly point-of-care biosensor has significant potential for providing a cost-effective alternative for dopamine detection in urine.