Macrophage migration inhibitory factor (MIF) is an important cytokine related to host defenses and autoimmune diseases. Here, we reported two full-length cDNA clones isolated from Chinese amphioxus (Branchiostoma belcheri tsingtaunese). Amino acid sequences analysis and structure prediction of these two molecules, called Bbt-MIF-I and Bbt-MIF-II, respectively, indicated that several conservative domains existed in the two amphioxus MIFs and their sequences were highly homologous to their counterparts of other species. Intriguingly, the Bbt-MIFs gene is present in multi-copy per haploid genome, which is very unusual compared with vertebrate's MIF gene given the known genome duplication theory. The genomic copy number, expression pattern of MIF gene and phylogenetic analysis of MIF proteins all suggested that a leap forward happened for MIF gene during the evolution from invertebrate to vertebrate. Considering the crucial role of MIF in innate immunity, MIF might serve as one of key molecular markers of evolution of immune system.