Ultrasound (US) molecular imaging provides a non-invasive way to visualize tumor tissues at molecular and cell levels and could improve diagnosis. One problem of using US molecular imaging is microbubbles challenges, including instability, short circulation time, and poor loading capacity and penetrability. It is urgent to design new acoustic contrast agents and new imaging methods to facilitate tumor-targeted imaging. In this study, phase-shift poly lactic-co-glycolic acid (PLGA) nanoparticles modified with folate as an efficient US molecular probe were designed and the long-term targeted imaging was achieved by low-intensity focused US (LIFU) irradiation.A new 5-step method and purification procedure was carried out to obtain uniform folic acid polyethylene glycol PLGA (PLGA-PEG-FA), the structure of which was confirmed by 1H nuclear magnetic resonance spectroscopy and thin-layer chromatography. Perflenapent (PFP) was wrapped in PLGA-PEG-FA by a double emulsion solvent evaporation method to obtain PFP/PLGA-PEG-FA nanoparticles. The targeted ability of the resulting nanoparticles was tested in vivo and in vitro. LIFU irradiation can irritate nanoparticle phase-shift to enhance tumor imaging both in vivo and in vitro.PLGA-PEG-FA was a light yellow powder with a final purity of at least 98%, the structure of which was confirmed by 1H nuclear magnetic resonance spectroscopy and thin-layer chromatography. Highly dispersed PFP/PLGA-PEG-FA nanoparticles with spherical morphology have an average diameter of 280.9±33.5 nm, PFP load efficiency of 59.4%±7.1%, and shells, thickness of 28±8.63 nm. The nanoparticles can specifically bind to cells expressing high folate receptor both in vivo and in vitro. Ultrasonic imaging was significantly enhanced in vitro and in vivo by LIFU irradiation. The retention time was significantly prolonged in vivo.Phase-shift PFP/PLGA-PEG-FA nanoparticles induced by LIFU can significantly enhance ultrasonic imaging, specifically targeting tumors expressing folate receptor. As a potential targeting acoustic molecular probe, PFP/PLGA-PEG-FA nanoparticles can be used to achieve targeted localization imaging.