For the bottom-up based stereolithography (SL) process, a separation process is required to detach the newly cured layer from the constrained surface in the fabrication process. Excessive separation force will cause damage to the built layers and the constrained surface. Different surface coatings, platform motions including tilting and sliding, and the utilization of oxygen-permeable films have been developed to address the separation-related problems. Among these approaches, the vibration-assisted (VA) separation method to reduce the separation force has limited study. The underlying mechanism of the VA separation-based method remains unexplored, and the best way to use VA separation in the bottom-up based SL process is still unclear. In this paper, a new VA separation design for the SL process is presented. A prototype system was built to study the VA separation mechanism. Experiments on the separation performance under different parameters, including vibration frequency, pre-stress level, and exposure area, were conducted. Based on the collected separation force data, an analytical model based on the mechanics of fatigue fracture was built. The separation behaviors related to different shape size and topology were also studied and compared. The results showed that the separation force in SL was significantly reduced using the VA separation-based method. Furthermore, the relationship between the separation force and the separation time conforms to the stress-based fatigue model. This study also provides insights on how to choose process parameters by considering the trade-offs between separation force and building efficiency.