Förster resonance energy transfer (FRET) is a powerful tool in biological research and has been widely used in the study of biomolecular interactions. SUMOylation is an important post-translational modification that is involved in many key biological processes. As a multi-step cascade reaction, SUMOylation involves multiple enzymes and protein-protein interactions. Here, we report the development of an in vitro FRET-based high-throughput screening (HTS) assay in SUMOylation. This assay is based on steady state and high efficiency of the fluorescent energy transfer between CyPet and YPet fused to SUMO1 and Ubc9, respectively. We optimized the assay and performed a small-scale pilot study to validate the screening platform. Carried out in 384-well plate format, our FRET-based HTS provides a powerful tool for large-scale and high-throughput applications.