MicroRNAs (miRNAs) as endogenous small non-coding RNAs play key regulatory roles in diverse biological processes via degrading the target mRNAs or inhibiting protein translation. Previously many researchers have reported the identification, characteristic of miRNAs and the interaction with its target gene. But, the study on the regulation of miRNAs to biological processes via regulatory the key signaling pathway was still limited. In order to comprehend the regulatory mechanism of miRNAs, two small RNA libraries from the spleen of miiuy croaker individuals with or without poly(I:C) infection were constructed. The 197 conserved miRNAs and 75 novel miRNAs were identified, and 14 conserved and 8 novel miRNAs appeared significant variations. Those differently expressed miRNAs relate to immune regulation of miiuy croaker. Furthermore, expressions of four differently expressed miRNAs were validated by qRT-PCR, and the result was consistent with sequencing data. The target genes of the differently expressed miRNAs in the two libraries were predicted, and some candidate target genes were involved in the RIG-I-like receptor (RLR) signaling pathway. The negative regulation of miRNAs to target genes were confirmed by comparing the expression pattern of miRNAs and their target genes. The results of regulating target genes were that firstly directly or indirectly activating the downstream signaling cascades and subsequent inducting the type I interferon, inflammatory cytokines and apoptosis. These studies could help us to deeper understand the roles of miRNAs played in the fish immune system, and provide a new way to investigate the defense mechanism of fish.