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The finger millet (Eleusine coracana (L.) Gaertn) genome, comprised 166 conserved microRNAs (miRNAs) belonging to 39 families and three novel miRNAs. The miR169 is one of the most conserved miRNA families, while Eco_N1 is a species-specific miRNA prevalent in finger millet. Its members regulate the expression of genes encoding the Nuclear Factor-Y subunit A (NF-YA) via transcript cleavage. However, the role of miRNA genes in regulating the expression of NF-YA transcription factors in finger millet needs to be deciphered. The present study characterized 166 conserved and novel miRNAs (Eco_N1, Eco_N2 and Eco_N3). Further, secondary structures were predicted, and the potential miR genes targeting the NF-YA transcription factors regulating abiotic stress tolerance were analysed. Twenty-three Eco-miR169 members and one Eco_N1 miRNA targeting EcNF-YA13 were identified in the finger millet genome. The presence of relevant cis-elements such as ABRE (abscisic acid-responsive elements), DRE (dehydration-responsive element), and MYB (myeloblastosis) indicates that the target of Eco-miR169 might be involved in abiotic stress responses. The tissue-specific RNA-seq transcriptomic expression pattern of Eco-miR169 showed variable fold of expression in seedlings compared to the control. At the same time, the expression of EcNF-YA13 (target genes of Eco-miR169 members and Eco_N1) presented a downregulated trend under salinity and dehydration conditions compared to the control. Tissue-specific RNA-seq followed by expression analysis confirmed the antagonistic effect of Eco-miR genes on EcNF-YA13. In a nutshell, the results of this study could be utilized as a platform for further exploration and characterization of finger millet Eco-miR169-EcNF-YA13gene regulatory network.