Toll-like receptors (TLRs) as important pattern recognition receptors, play critical roles in identifying pathogens and activating the immune response. However, when the dysregulation was occurred in this process, it could lead to excessive immune response, so it need many regulatory factors to control this process. Recently, microRNAs (miRNAs) have been shown to act as an important regulator in TLRs signaling pathway. As a member of TLRs family, TLR28 has been newly discovered in teleost fish, and play an important role in the immune response. In this study, we found that the expression of miR-21 was up-regulated after poly(I:C) stimulation, and miR-21 could inhibit the expression of cytokines. Then we predicted the target genes of miR-21, and found that TLR28 is a direct target of miR-21, which could be significantly down-regulated by both miR-21 mimics and pre-miR-21. These results suggested that miR-21 can inhibit the expression of cytokines by negative regulation of TLR28, thereby inhibiting the generation of excessive immunity and maintaining the balance of the body. This study is the first to demonstrate that miRNA can suppresses cytokines by regulating the TLR signaling pathway in teleost fish, and also can provides some new ideas for the research of the regulation of miRNA and immune system in mammals.