The gene regulating flavonoid glycosylation modification in Eurotium cristatum (EcUGT88E3) was cloned and a prokaryotic expression vector was constructed for in vitro expression, which can provide a foundation for further functional validation.The homologous sequence cloning technique was used to clone the EcUGT88E3 gene. Following a bioinformatics analysis, an expression system comprising the pET28a-EcUGT88E3/BL21(DE3) was constructed and the induction conditions were optimised. Ultimately, this enabled the heterologous expression and purification of the EcUGT88E3 gene.Results indicate that the full-length EcUGT88E3 gene spans 1704 bp, including 75 bp of 5′-UTR, 207 bp of 3′-UTR, and 1422 bp open reading frame encoding 474 amino acids. Bioinformatics prediction suggests the encoded protein is hydrophobic with a relative molecular mass of 52.03 ku. Protein structure prediction indicates that EcUGT88E3 lacks signal peptide and transmembrane domain features, classifying it as a microsomal-targeted protein containing GT1 and GT-B superfamily domains. The recombinant EcUGT88E3 protein was expressed as inclusion bodies, with a molecular weight consistent with predictions. The optimal induction conditions for the pET28a-EcUGT88E3 prokaryotic expression vector in E. coli BL21 (DE3) were 27°C and 0.2 mmol/L IPTG for 4 hours.