Covalent attachment of ubiquitin onto lysine residues of the substrate requires the coordinated action of three classes of enzymes： the E1 ubiquitin-activating enzymes， the E2 ubiquitin-conjugating enzymes， and the E3 ubiquitin ligases. These ubiquitination-related proteins play a pivotal role in plant development and plant physiology.The APPBP1/UBA3 （E1）， UBC12 （E2）， Cullin1-Rbx1 （E3） and Nedd8 were used to reconstitute SCF E3 ligase activity in vitro to study protein ubiquitination in vitro. Ub （Ubiquitin） labeled with FITC-Cysteine green fluorescein was prepared. The ubiquitination system of SCF ubiquitin ligase in vitro was constructed. The self-ubiquitination modification of Cullin1 protein in SCF ubiquitin ligase was rapidly detected. The results showed that the autoubiquitination active reaction system of SCF E3 in vitro established has high operability and convenience.