In the present study, full length cDNA sequence of the major histocompatibility complex II invariant chain (MHC II) Ii gene was cloned in Platichthys stellatus by using 3'RACE and 5'RACE technique, and the sequence and structural analysis of the MHC II Ii gene by using bioinformatics method. The results showed that the MHC II Ii cDNA sequence consisted of 1766 bp, including 135 bp at 5' region, 794 base pairs at 3'untranslated region and an open reading frame with 837 bp which could encode 279 amino acids. The theoretical molecular weight was 30.848 kDa, pI was 6.89. Comparison of the amino acid sequences and phylogenetic analysis showed that the P. stellatus MHC II Ii had the close relationship with Dicentrarchus labrax, Lutjanus argentimaculatus and Siniperca chuatsi. The results of different tissues indicated that the expression levels of MHC II Ii mRNA were higher in spleen and head kidney, lower in gill, blood, muscle, heart and gonads by Real-time Quantitative PCR technique. After pathogen infection, the expression levels of immune spleen and head kidney increased most significantly, and liver and intestinal expression levels increased slightly. This study can provide a theoretical basis for the function and regulation mechanism of P. stellatus MHI, and for basic research of marine fish disease resistance breeding.