MicroRNAs are endogenous highly conserved short (~ 21 nucleotides) non-coding RNA molecules that play key roles in post-transcriptional gene regulation by translational inhibition or by target mRNA cleavage. In this report, using high stringent computational-based methods, a total of 101 putative miRNAs were identified from European sardine fish (Sardina pilchardus Walbaum, 1792). All the precursors of identified sardine miRNAs formed stable stem-loop structures and displayed high minimum free energy index (MFEI) values. For the experimental validation of the computationally predicted miRNAs, a tissue-specific quantitative study of eight randomly selected putative sardine miRNAs (spi-miR9, spi-miR26, spi-miR128, spi-miR129, spi-miR132, spi-miR212, spi-miR219, and spi-miR338) was performed in brain and liver and all the selected miRNAs were found to be overexpressed in brain tissue. Moreover, using RNAhybrid, a total of 83 potential target proteins of the characterized sardine miRNAs were identified those are involved in transcription, cellular development, defense mechanism, and various signaling pathways. To the best of our knowledge, this is the first report of sardine microRNAs and their targets.