We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin–biotin linkage to form the PS–apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS–apt15–th complex. We demonstrated that the PS–apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS–apt15–th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS–apt15–th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.