Methods for the isolation and analysis of extracellular vesicles (EVs) have been extensively explored in the field of life science and in clinical diagnosis in recent years. The separation and efficient recovery of high-purity target EVs from biological samples are important prerequisites in the study of EVs. So far, commonly used methods of EV separation include ultracentrifugation, filtration, solvent precipitation and immunoaffinity capturing. However, these methods suffer from long processing time, EV damage and low enrichment efficiency. The use of acoustophoretic force facilitates the non-contact label-free manipulation of cells based on their size and compressibility but lacks specificity. Additionally, the acoustophoretic force exerted on sub-micron substances is normally weak and insufficient for separation. Here we present a novel immuno-acoustic sorting technology, where biological substances such as EVs, viruses, and biomolecules, can be specifically captured by antibody/receptor coated microparticles through immunoaffinity, and manipulated by an acoustophoretic force exerted on the microparticles. Using immuno-acoustic sorting technology, we successfully separated and purified HER2-positive EVs for further downstream analysis. This method holds great potential in isolating and purifying specific targets such as disease-related EVs from biological fluids and opens new possibilities for the EV-based early diagnosis and prognosis of diseases.