Thirty Male of C57BL/6NTac mice were used for this study. 10 Obese, 10 diabetic and 10 control. Total RNAs were extracted from the serum and pooled for each of target groups of animals. Next, the small RNAs were sequenced using the TruSeq small RNA Library Prep Kit in a MiSeq Illumina sequencer. Individual validation of the twenty microRNAs selected as favourable biomarkers was carried out using RT-qPCR. A bioinformatics-pipeline was established to distinguish 1,915 from the large pool of sequencing data. By using the comparison of the log2 fold change of obese and diabetic (with normal mice as control; FC ≥ 2) Only 127 Mature miRNAs were used for statistical analysis, the remaining 1,788 were excluded. Interestingly, 17 miRNAs were found specifically in the obese group in conjunction to the 26 miRNAs in the particular diabetic group. From the obese and diabetic groups, miR-144, miR-181, miR-27, miR-191, miR-128, miR-221 showed up-regulated expressions. From the diabetic group, miR-144-3p, miR-451, miR-328 and miR-142 displayed down-regulated expressions, whereas, the obese group did not illustrate any significant down-regulated miRNAs. These were confirmed through RT-qPCR analysis