As a key enzyme downstream of anthocyanin metabolism pathway, dihydroflavonol 4reductase plays an important role in regulating anthocyanin synthesis. In this study, a DFR gene was successfully cloned by RTPCR method with Ophiorrhiza japonica as materials. Subsequently, the properties of OjDFR3 protein were analyzed through bioinformatics methods. Meanwhile, the prokaryotic expression vector of OjDFR3 was constructed and its recombinant protein was prepared and purified which would lay a foundation for the further study on OjDFR3 function as well as the synthesis and regulation of anthocyanins in O. japonica. The results showed that: (1) a DFR gene was successfully cloned (OjDFR3), and sequence analysis displayed that the fulllength cDNA of OjDFR3 was 1071 bp, encoding 356 amino acids, and the putative protein molecular weight was 39.52 kD; (2) Bioinformatics analysis showed that the protein encoded by OjDFR3 gene was composed of 20 kinds of amino acids, of which leucine was the most. OjDFR3 was a hydrophilic protein without signal peptide, and was likely located in cytoplasm. Simultaneously, structure prediction showed that OjDFR3 was composed of αhelix, extended chain and irregular coil; (3) Prokaryotic expression analysis exhibited that the recombinant plasmid pET32aOjDFR3 could be expressed in E. coli BL21(DE3), and the optimal expression conditions were 37 ℃, 4 h, IPTG concentration of 0.8 mmol/L. Meanwhile, the purity of recombinant protein was best at 100 mmol/L and 200 mmol/L imidazole concentrations. (4) According to the optimal conditions, a large number of proteins with good concentration and purity were obtained. In conclusions, the results of this study will lay a foundation for further study on the function of this gene as well as the synthesis and regulation of anthocyanin in O. japonica.