MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at a post-transcriptional level. Their tissue-specific expression patterns, high stability in blood, and ability to cross the blood-brain barrier make them promising biomarker candidates for diagnosing central nervous system (CNS) disorders. However, further characterization of miRNA biomarkers is needed to identify key factors for diagnosing CNS disorders. In this study, we aimed to detect and characterize a differentially expressed miRNA as a biomarker for Major Depressive Disorder (MDD). We performed RNA sequencing on peripheral blood samples obtained from control and MDD patients to identify differentially expressed miRNAs in MDD. We identified a number of differentially expressed candidates and further filtered them to a miRNA candidate enriched in the CNS and expressed in both humans and mice. After filtering, we selected a miRNA which is overexpressed in MDD patient blood samples for further analysis. We demonstrated its specificity to neurons in the CNS via RT-qPCR. To mimic the expression pattern observed in MDD, we overexpressed the candidate miRNA in neurons and measured neuronal plasticity and activity. Overexpression of the candidate miRNA resulted in lower neural activity in vitro. In conclusion, we have identified a miRNA that affects CNS neuron activity and can serve as a blood-based biomarker candidate for diagnosing MDD.