Toll-like receptors (TLRs) are a group of pattern-recognition receptors which play vital roles in ligand recognition and activation of the innate immune response. As an important member of TLRs family, TLR1 is mainly responsible for PAMPs from bacteria and play a pivotal role in sensing microbial products. Recent studies revealed that TLR1 could perceive LPS stimulation and transfer signals to activate the NF-κB pathway, whereas ligands and signaling pathway of TLR1 are still unclear in fish. Growing evidence has shown that miRNAs (microRNAs) play as negative regulators in controlling the diverse of biophysical and biochemical processes at the post-transcriptional level. In this study, we used a combination of bioinformatics and experimental techniques to exhibit that both miR-8159-5p and miR-217-5p were the direct negative regulators of TLR1 in miiuy croaker. Furthermore, dual-luciferase reporter assays showed that combining miR-8159-5p and miR-217-5p exhibited a greater negative regulatory effect on TLR1 than only miR-8159-5p or miR-217-5p. Additionally, we also demonstrated that the expression of both the two miRNAs could be up-regulated by LPS stimulation in either LPS-stimulation spleen tissue or LPS-treated cultured macrophage, which indicating that miR-8159-5p and miR-217-5p could be induced by LPS and may be as the negative regulators of TLR1 involved in the immune response to LPS stimulation. These results would enhance our understanding of the miRNA regulation in fish TLR signaling pathways.