Protein SUMOylation is a key modification for regulating the fate of proteins and it is widely involved in plant development and stress responses. The SUMO molecules are conjugated to the lysine residues of substrate proteins via isopeptide bonds by enzyme reaction. SUMOylation is mediated by an enzyme cascade composed of a SUMO activating enzyme complex (E1), a SUMO-conjugating enzyme (E2) and usually a SUMO ligase (E3). Here, we report an efficient in vitro detection system for SUMOylation of plant proteins. We established a system for SUMOylation detection of plant proteins by reconstructing the Arabidopsis SUMOylation enzyme cascade in Escherichia coli. Using this system, SUMOylation of several substrates were detected via immunoblotting. Therefore, this system simplifies the SUMOylation detection of plant protein substrates and provides a powerful tool for functional analysis of SUMOylation in plant cells.