Exosomes of cancer cells play an important role in the proliferation, adhesion, and migration of tumors. Especially, exosomes in the tumor microenvironment can reflect the proliferation of tumors directly, thus serving as ideal referenced markers of the possibility and grade of malignancy in neoplasms. However, the sensitive and accurate detection of exosomes remains challenging. In this work, a novel three-dimensional (3D) plasmonic nanostructure was constructed for exosomal miRNA detection. It combined the advantages of Au nanostar monolayer and Ag nanowire monolayer to provide multiple hot spots. Moreover, Au nanostar monolayer changed the isotropic electrochemiluminescence (ECL) into polarized emission. The Ag nanowire monolayer worked as waveguides for the light direction. As a result, the polarized resolution and intensity of ECL signal were improved. The polarized ECL emission was significantly increased by 47.1 times. This high-resolution polarized ECL sensor was used for detecting exosomal miRNA-146b-5p in the thyroid tumor microenvironment. This sensor showed the linear range from 1 fM to 1 nM with a detection limit of 0.3 fM. The satisfactory results indicated the developed 3D plasmonic nanostructure-based ECL sensor had great potential in biosensing and clinical diagnosis.