Abstract In this study, a novel emulsion diffusion method was used to prepare enteric Eudragit L100-55 nanoparticles by ultrasonic dispersion and diffusion solidification. Omeprazole was selected as the model drug. The prepared nanoparticles were in spherical shape and exhibited negative zeta potential. The Fourier transform infrared spectroscopy results indicated that no molecular interaction occurred between the drug molecule and polymer chain. In addition, the nanoparticles showed a strong pH-sensitive release in vitro. A mild cytotoxicity of nanoparticle was observed in the subsequent studies, and the particle cellular uptake study showed that the nanoparticles could be taken up by Caco-2 cells after 0.5h incubation. Our results indicated that the enteric Eudragit L 100-55 nanoparticle could be synthesized successfully via this ultrasonic solidification method, which also could be applied to prepare other pH-sensitive polymer nanoparticles.