The present study aimed to prepare perfluoropropane (C3F8)-filled poly(lactic-co-glycolic acid) (PLGA) nanoparticles and investigate the feasibility of using PLGA nanoparticles as an ultrasound contrast agent. The PLGA nanoscale ultrasound contrast agent was prepared using a modified double-emulsion solvent evaporation method. Camphor in the form of a sublimable porogen was added to render the nanoparticles hollow and enable C3F8 gas introduction. Various physicochemical properties of PLGA nanoparticles, including morphology, size and dispersion, were analyzed by electron microscopy and dynamic laser scattering. In vitro ultrasound imaging of C3F8-filled PLGA nanoparticles was also investigated under various imaging conditions. Further in vivo ultrasound imaging was conducted on male rats following intratesticular injection of PLGA nanoparticles. C3F8-filled PLGA nanoparticles with a mean diameter of 152.0±58.08 nm were obtained. Electron microscopy revealed spherical-shaped nanoparticles with smooth surfaces, a capsular morphology and a large hollow within. In vitro ultrasound imaging of hollow PLGA nanoparticles indicated marked signal enhancement. Local intensity of the acoustical signal continued to increase during PLGA-nanoparticle injection into the testicle and the ability of hollow PLGA nanoparticles to enhance ultrasound imaging in vivo was demonstrated. The enhancement image of testicular tissue following injection with C3F8-filled PLGA nanoparticles was sustained for a minimum of five minutes. In conclusion, the hollow C3F8-filled PLGA nanoparticles were demonstrated to have potential for applications as a novel ultrasound contrast agent.