Circulating microRNAs (miRNAs) are emerging biomarkers for type 2 diabetes mellitus (T2DM). However, a comprehensive characterization of the serum miRNA profile in patients with T2DM-associated microvascular disease (T2DMC) has rarely been reported. In this study, we obtained serum samples from 184 T2DM patients (92 with microvascular complications and 92 free of complications) and 92 age/gender-matched controls. The levels of 754 miRNAs were initially analyzed using a TaqMan Low Density Array (TLDA) in three pooled samples from 24 T2DM patients, 24 T2DMC patients and 24 controls. Markedly upregulated miRNAs in the patients' groups were subsequently validated individually by quantitative reverse-transcription PCR (RT-qPCR) in the same samples used for TLDA and further confirmed in another larger cohort consisting of 68 patients with T2DM, 68 patients with T2DMC and 68 controls. Five miRNAs were significantly upregulated in T2DM patients (p < 0.05) including miR-661, miR-571, miR-770-5p, miR-892b and miR-1303. Moreover, the levels of the five miRNAs were higher in patients with complications than in those without complications. Regression analyses revealed the five miRNAs were significantly correlated with microvascular complications (p < 0.05). The five serum miRNAs identified in our study hold potential as auxiliary biomarkers and novel risk factors for T2DM-associated microvascular complications.