High-molecular-weight polymeric nanoparticles are critical to increasing the loading efficacy and tuning the release profile of targeted molecules for medical diagnosis, imaging, and therapeutics. Although a number of microfluidic approaches have attained reproducible nanoparticle synthesis, it is still challenging to fabricate nanoparticles from high-molecular-weight polymers in a size and structure-controlled manner. In this work, an acoustofluidic platform is developed to synthesize size-tunable, high-molecular-weight (>45 kDa) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) nanoparticles without polymer aggregation by exploiting the characteristics of complete and ultrafast mixing. Moreover, the acoustofluidic approach achieves two features that have not been achieved by existing microfluidic approaches: (1) multi-step (≥2) sequential nanoprecipitation in a single device, and (2) synthesis of core-shell structured PLGA-PEG/lipid nanoparticles with high molecular weights. The developed platform expands microfluidic potential in nanomaterial synthesis, where high-molecular-weight polymers, multiple reagents, or sequential nanoprecipitations are needed.