Tachypleus tridentatus belongs to the arthropod phylum, and its blood contains a lot of hemocyanin. Hemocyanin plays an important role in the organism, such as transporting oxygen, immune defense, and energy storage. Hemocyanin also has many applications in vitro, such as being made into biosensors and immune products. This study aims to obtain high-purity hemocyanin of T. tridentatus and explore its potential application value. In this study, hemocyanin was extracted from hemolymph of T. tridentatus and purified with AKTA protein purification system. The purified hemocyanin was qualitatively analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology. Finally, 22 amino acid sequences of hemocyanin from 5 species were downloaded from the uniprot protein database, then the N-J phylogenetic tree was constructed. The results showed that the purity of the purified hemocyanin was high. Twelve types of hemocyanin subunits were identified from hemocyanin of T. tridentatus, including Hemocyanin subunit Ⅰ, Hemocyanin subunit Ⅱ, Hemocyanin subunit Ⅲa, Hemocyanin subunit Ⅲb, Hemocyanin subunit Ⅳ, Hemocyanin subunit Ⅴ, Hemocyanin subunit Ⅵ, etc. According to the uniprot database, these subunits were first found in Carcinoscorpius rotundicauda and Limulus polyphemus. The N-J phylogenetic tree shows that hemocyanin of Tachypleus tridentatus has a high degree of homology with C. rotundicauda and L. polyphemus. It was also found that there was a close evolutionary relationship of hemocyanin between hemocyanin of Limulidae and Litopenaeus vannamei and Scylla serrata, which suggests that the function of hemocyanin in T. tridentatus can be inferred from the function of hemocyanin in other species. This study obtained high-purity hemocyanin from hemolymph of T. tridentatus, and the results of this study are helpful to the function prediction and application research of Chinese horseshoe crab hemocyanin.