For the bottom-up based stereolithography (SL) process, a separation process is required to detach the newly cured layer from the constrained surface in order to accomplish the fabrication of the current layer. 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 proposed to address the separation-related problems. Among these approaches, there is a limited study on the vibration-assisted (VA) separation method to reduce the separation force. The underlying mechanism of the VA-separation-based method remains unexplored for the SL-related additive manufacturing, 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 bottom-up SL process is presented. A prototype system has been built to study the VA separation mechanism in SL. 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 sizes and topologies 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 well. This study provides insights on how to choose process parameters by considering the trade-offs between separation force and building efficiency.