The total RNA extracted from the hemocyte sample of Portunus trituberculatus was used to amplify the DNA sequence encoding an open reading frame for C-type lectin like-domain protein (noted CTLD) by RT-PCR. The sequence was then cloned into pMD18-T vector and was sequenced. The recombinant plasmid was digested by BamH I and Xho I. The target gene was subsequently inserted into the pET-32a(+) vector，which was also digested with the corresponding restriction endonuclease. The recombinant plasmid p32a-CTLD was transformed into Escherichia coli BL21(DE3) and then induced by isopropylthio-β-D-galactoside (IPTG) .The fusion protein was purified through His-Band resin chelating chromatography and Amicon Ultra centrifugal filter devices，and agglutination activity was assayed finally. Results showed that the CTLD recombinant plasmid was successfully obtained by double enzyme identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and western-blot analysis showed that the fusion protein was successfully expressed. The fusion protein of about M39600 was successfully induced, which was identified by SDS-PAGE and western blot analysis. For the agglutination assay, no obvious agglutination was detected on the rabbit erythrocytes, while the agglutination titer of the mice erythrocytes was 22. No obvious agglutination was observed when Vibrio alginolyticus，V. harueyi ，V. anguillarum， Staphylococcus aureus were incubated with the recombinant protein. However, mass agglutination was observed after E. coli was incubated with the recombinant protein for 1h. This study provides a good foundation for further studies on the function of C-type lectin.