Alzheimer's Disease (AD) is a complex and progressive neurodegenerative disorder that affects millions of people worldwide. However, current treatments and diagnostic methods are ineffective in early detection and intervention. Recently, researchers have focused on the dysregulation of small non-protein-coding microRNA (miRNA) and the associated post-transcriptional gene alteration in AD. Biofluids, such as serum and cerebrospinal fluid, contain miRNAs that can be identified and quantified. The project aims to identify circulating miRNAs within serum and CSF, which can be used as non-invasive diagnostic biomarkers that facilitate the early detection of disease, the identification of drug targets, and potentially the continual monitoring of disease progression. To achieve this, 40 patients, including 20 clinical AD and 20 healthy controls, were recruited at the Neurology Unit, Hospital of Brescia, Italy. Plasma samples were obtained simultaneously with the lumbar puncture, and total RNA was isolated for miRNA sequencing. RNA sequencing analysis in serum and CSF samples allowed us to identify some differentially expressed miRNAs. The significant miRNAs identified in the sequencing were validated using quantitative PCR, and the miRNA significantly modulated in the first cohort of patients was investigated in a separate cohort of 80 other patients. Validated miRNAs regulate essential pathology pathways, making them therapeutic targets. Such information may not only support disease diagnosis but also provide the opportunity to evaluate therapeutic interventions earlier in the disease process. Overall, this study highlights the potential of miRNAs as non-invasive biomarkers and helps identify new therapeutic targets.