Myeloid differentiation factor 88 (MyD88) is an important adaptor protein in the TLR signaling pathways. In the present study, we firstly cloned and characterized Miichthys miiuy MyD88 (Mimi-MyD88) cDNA and gene. The Mimi-MyD88 gene was 3,470 bp consisting of five exons and four introns. The cDNA was composed of 1,627 bp with an 867-bp open reading frame encoding a polypeptide of 288 amino acid residues. The theoretical molecular mass and isoelectric point of this polypeptide were 33.25 and 4.96 kDa. Comparison of the deduced amino acid sequence showed that the conserved death domain and the typical Toll/IL-1 receptor domain are very similar to those presented in other mammals, amphibians, and fishes. To identify potential role of MyD88 in fish innate immunological surveillance, the constitutive Mimi-MyD88 mRNA is detected by real-time quantitative polymerase chain reaction. Results demonstrated that Mimi-MyD88 is broadly expressed in ten normal tissues, with the lowest expression was observed in kidney and the highest was in liver. The transcriptional expression also revealed that Mimi-MyD88 was significantly up-regulated in liver, kidney, and spleen after challenge by Gram-negative bacteria, Vibrio anguillarum. Via contrasted the expression of MyD88 and TLR2 in kidney, we evaluated TLR2 plays an indispensable role in MyD88 transcription, but not absolutely dominant. The combined expression still indicated that MyD88 plays a universal role in keeping immune surveillance for pathogens. Phylogenetic analysis suggested that Mimi-MyD88 gene is classified into the piscine cluster and most closely related to large yellow croaker Larimichthys crocea.