Ropivacaine loaded microspheres offer an attractive alternative for prolonging the duration of local anesthetics for the pain management. However, traditional microcapsulation methods could not provide microspheres with desired drug loading efficiency and a narrow size distribution, leading to poor patient compliance as well as preparation repeatability. In this study, we proposed to combine O/W emulsion method with novel premix membrane emulsification technique. After the optimization of fabrication procedure, ropivacaine loaded microspheres with narrow size distribution (span 0.469), high drug loading efficiency (49%) and an ideal constant release behavior have been obtained. Especially, it was found that solidification process affected the internal structure of microspheres and the physical state of encapsulated ropivacaine. Furthermore, it was interested that lower polymer molecular weight resulted in higher encapsulation efficiency, which was not consistent with reported results. It was found that this was ascribed to the interaction of PLGA-ropivacaine. All these aspects had considerable influences on the characteristics of microspheres. In addition, the cytotoxicity on different cell lines (SHSY5Y and HaCaT cells) was detected qualitatively and quantitatively. It showed that ropivacaine loaded microspheres did reduce the Reactive oxygen species (ROS) and cytotoxicity prominently compared to free ropivacaine, which could be attributed to the sustained and steady drug release behavior. This study provided some pioneering ideas for encapsulating small molecule drug with poor solubility. The optimized microspheres could be chosen as an ideal candidate for the ropivacaine sustained release with its better drug adherence, lower toxicity of drug burst release and best therapeutic effect.