Background: Alterations in high-density lipoprotein (HDL) subfractions, especially in the HDL2b-subfraction, have been reported in type 2 diabetes mellitus (T2DM). However, new methods for convenient and reliable quantitation of HDL2b are yet to be developed. Methods: Thirty-eight patients with T2DM were enrolled and age-, sex-and body mass index (BMI)-matched controls were selected from the same population. A microfluidic chip method was employed to analyse serum HDL subfractions. Results: The microfluidic chip method revealed a significant reduction in HDL2b and its ratio to total HDL in T2DM patients. There was a reverse correlation for total HDL and HDL2b, and its ratio with triglycerides, homeostasis model assessment-insulin sensitivity index (HOMA-IS) and insulin resistance index (HOMA-IR). Conclusions: We have shown a reduction of HDL2b and its ratio to total HDL by a novel chip method in T2DM patients. The significant correlation between HDL2b and HOMA-IS and HOMA-IR may have further predictive value in clinical utility.