Type 2 diabetes mellitus (T2DM) has emerged as a global epidemic issue, with high rates of disability and fatality. Traditional diagnostic biomarkers are typically detected once a metabolic imbalance has already occurred, thus the development of early diagnostic biomarkers is crucial for T2DM. Metabolomics studies have identified several predictive biomarkers for T2DM, including miR-320. Our previous research found that miR-320b was significantly downregulated in T2DM patients, but the underlying mechanism remains unclear. Therefore, this study was designed to investigate the significance of miR-320b for T2DM diagnosis and to explore the involved molecular mechanism. A total of 50 patients with T2DM and 80 sex- and age-matched healthy subjects were selected. The plasma miR-320b of all participations was detected by qRT-PCR and its correlations with other biomarkers of T2DM were analyzed. Besides, the expression of miR-320b in HepG2 cells was suppressed by miRNA inhibitors. Then the glucose consumption of HepG2 cells was measured. The target gene of miR-320b was predicted by four bioinformatics tools and intersected these prediction results by Venny method. The T2DM relevant target genes were identified by the GeneCards database. To ensure disease relevance, these T2DM relevant target genes were subsequently intersected with the target genes of miR-320b. Protein-protein analysis (PPI) was used to screening the gene with the most connections in these target genes. Finally, the target gene of miR-320b specific to T2DM was confirmed directly by luciferase reporter assay. The expression of target gene in HepG2 cell culture supernatant and plasma of all participations was detected. Our results showed that the expression level of miR-320b was significantly lower in T2DM patients compared to the healthy controls. It was negatively correlated with fasting plasma glucose (FPG), glycated hemoglobin (HbA1C), and homeostasis model assessment of insulin resistance (HOMA-IR), but positively with HOMA-. The glucose consumption of HepG2 cells in the miR-320b inhibitor group was significantly lower compared to inhibitor-NC and blank control group. We predicted and confirmed that phosphatase and tensin homolog (PTEN) was the direct target gene of miR-320b using Bioinformation tools and luciferase reporter assay. Moreover, the concentration of PTEN was significantly higher in the HepG2 cell culture supernatant and plasma of T2DM patients. Our research demonstrated a negative correlation between miR-320b and FPG, HbA1C, and HOMA-IR, while exhibiting a positive correlation with HOMA-. Suppressing miR-320b expression would impair glucose consumption of HepG2 cells through PI3K pathway by targeting PTEN. These results suggest that miR-320b may be a potential biomarker for diagnosing T2DM and a promising target for therapeutic intervention.