The Y-box protein family is a highly conserved nucleic acid binding proteins which are conserved from bacteria to human. The new member of this family from Strongylocentyotus intermedius were isolated and characterized. The full-length sea urchin Y-box cDNA was 2425bp in length and contained a 996bp open reading frame (ORF), a 1348 bp 3 UTR and a 81 bp 5 UTR. The full-length Y-box cDNA had AATAA special sequences in the 3′ UTR and polyA regions. The full-length Y-box cDNA encoded 331 amino acids constituting a 37.4KD protein molecule with an isoelectric point of 8.55. The precursor protein was hydrophily. The Y-box protein contain three protein domain involved N-glycosylation site, hydrophily C-tail end and cold shock domain CSD, without transmembrane. CSD area in Y-box protein family was very conservative .The secondary structure of the precursor protein consisted primarily of a 1.98% alpha helix, with a 9.97% strand chain and random coil 88.22%, without β folding. The comparison revealed that the similarity of the Y-box precursor protein ranged from 22.37% to 41.26% with other animals. Y-box protein secondary structure is very similar with human and zebrafish. The evolutionary tree indicated that the Y-box precursor protein was clustered with Aplysia californica、Chlamys farreri and Ciona intestinalis. Xenopus tropicalis、Gallus gallus、Mus musculus、Homo sapiens、Lethenteron camtschaticum and Carassius auratus belong to vertebrates. Chironomus tentans and Drosophila virilis belong to land vertebrates. The Y-box gene expression pattern was detected in coelomic fluid, tube feet, peristomial membrane, male gonads, female gonad, muscle and intestinal tissue. The results in sea urchin Strongylocentrotus intermedius indicated that the difference was not significant. The expression of Y-box gene under temperature stress was reduced, and the expression quantity of Y-box gene decreased gradually with the increase of temperature. The results provide a reference for investigating the function of this gene.