In order to explore the molecular characteristics and expression characteristics of the rare minnow MHC Ⅱβ gene, the 810 bp cDNA sequence of the rare minnow Gobiocypris rarus MHC Ⅱβ was obtained by PCR amplification technology, including an open reading frame (ORF) of 759 bp, encoding 252 amino acids (aa). Bioinformatics analysis showed that there were four conserved cysteine residues and a GXXGXXXGXXXXXXG structure in the amino acid sequence of MHC IIβ, and the consistency with other relative fish was 51.78%-80.56%. The encoded protein molecules included a signal peptide, a MHC IIβ (β-1) domain, a IGc1 (β-2) domain and a transmembrane helical region. Quantitative Real-time PCR (qRT-PCR) results showed that the expression of MHC Ⅱβ gene was the highest in spleen, and higher in head kidney, gill and skin. After artificial infection with pathogenic bacteria, Yersinia ruckeri, the expression of head kidney was significantly up-regulated at 6 h, and liver was significantly up-regulated at 12 h, skin was significantly up-regulated at 24 h and 48 h, gill was significantly up-regulated at 6 to 24 h, and spleen was significantly down-regulated at 6 h, and approached the expression level of the control group at 96 h. The results show that MHC IIβ plays an important role in immune response, which provides reference for further revealing the regulatory mechanism of MHC family immune defense.