Background: Circulating miRNAs hold promise as biomarkers for understanding human diseases, including type 2 diabetes (T2D) and its vascular complications. However, conflicting findings in miRNA expression patterns exist, partly due to methodological variations in normalization procedures and study population heterogeneity. This cross-sectional study aimed to identify potential biomarkers for predicting and diagnosing T2D by analyzing differential expression patterns of circulating miRNAs in serum samples from patients with impaired fasting glucose (IFG) and new-onset T2D compared to euglycemic controls, using a commercial, array-based, high-throughput profiling system and real-time PCR. Methods: Thirty participants, aged 45-65 years, were categorized into three matched groups (n=10 each) based on glycometabolic status. Exclusion criteria included factors influencing miRNA levels (i.e., inflammation, obesity, smoking, pharmacological medications) and significant hypertriglyceridemia. Circulating miRNAs were extracted and profiled using real-time PCR on a high-throughput 384-well array, containing spotted forward primers for 372 miRNAs with known detectable expression in the human serum (QIAGEN). Data were analyzed with the GeneGlobe software and normalized by the global Ct mean method. Results: Among 372 analyzed miRNAs, 33 exhibited significantly altered expression in IFG and new-onset T2D compared to euglycemic controls, with 2 down-regulated and 31 up-regulated. Stringent analysis conditions, using a differential fold regulation threshold ≥10, revealed that nine miRNAs (miR-3610, miR-3200-5p, miR-4651, miR-3135b, miR-1281, miR-4301, miR-195-5p, miR-523-5p, and let-7a-5p) showed specific upregulation in new-onset T2D compared to IFG, indicating their potential as early biomarkers for T2D progression. Moreover, by conventional fold regulation thresholds of±2, miR-146a-5p was down-regulated and miR-1225-3p up-regulated exclusively in new-onset T2D patients. Bioinformatic analysis suggests a potential role of miR-1225-3p in T2D pathogenesis through interaction with MAP3K1 and HMGA1. Conclusions: This study contributes to the identification of circulating miRNAs as potential novel biomarkers for early T2D diagnosis. The findings provide a proof of concept for future mechanistic investigations, shedding light on the pathophysiology of T2D and its progression.