MicroRNAs (miRNAs) constitute a group of small non-coding RNAs (~22 nucleotides) and one of their main functions is to regulate the immune responses. Gram-positive bacterium, Streptococcus parauberis is the main causative agent of “Streptococcosis” in wide range of fish species. In this study, we performed high throughput sequencing analysis to identify the miRNA profile against S. parauberis infection in the spleen of zebrafish (Danio rerio). Overall, 349 known and 151 novel miRNAs were discovered. Among them, 12 known miRNAs (dre-miR-34b, dre-miR-135a, dre-miR-200b-5p, dre-miR-146b, dre-miR-31, dre-miR-17a-3p, dre-miR-222a-3p, dre-miR-731, dre-miR-301b-3p and dre-miR-30a-3p) and 9 novel miRNAs were differentially expressed (DE) in the spleen of S. parauberis challenged zebrafish. The identified 12 DE miRNAs were predicted to regulate 721 target genes. We confirmed the miRNA expression results by validating selected known and novel DE miRNAs using qRT-PCR. Gene Ontology (GO), Kyoto Encyclopedia of Genes (KEGG) pathway analysis and miRNA-mRNA interactions implies that specific target genes of DE miRNAs are associated with immune responses. The enriched pathways included Toll-like receptor (TLR), C-type lectin, NOD-like receptor, and RIG-I-like receptor signaling pathways, etc. Especially, dre-miR-200b-5p, dre-miR-146b, dre-miR-731, dre-miR-222a-3p, and dre-miR-34b were able to target potential immune-related genes such as il10, irak1, traf6, hspa8 and ikbke upon S. parauberis challenge. Thus, overall results could lay a foundation to understand the underlying immune regulatory role of miRNAs in response to pathogenic S. parauberis infection in teleosts.