A novel flexible test strip has been developed for the selective and sensitive detection of dopamine (DA) using an electrospun polyacrylonitrile nanofiber membrane as a substrate and doxorubicin (DOX)-functionalized graphene oxide (GO) as a fluorescent ligand. Employing the Langmuir-Blodgett technique, the fluorescent probe of GO/DOX was controllably and flatly applied onto the surface of nanofiber membranes. The adsorption behavior of DA and DOX on GO in solution and on the surface of solid test strips has been investigated, respectively. The results showed that DA exhibited a higher affinity for GO, both in solution and on the surface of test strips, compared to DOX. Due to the competitive adsorption of DOX and DA onto GO, the addition of DA to the GO/DOX complex led to a significant fluorescence enhancement, attributed to the replacement of the GO-supported DOX with DA of a higher adsorption affinity to GO, enabling the detection of DA. This nanofiber-based method proves to be simple, sensitive, selective, and effectively minimizes interference from common biomolecules and small molecules in serum. Thus, this portable, cost-effective, and selective nanofiber strip holds great promise for early of diseases DA levels.