Free fatty acids (FFA) as factors causing insulin resistance and dyslipidemia represent an important link between obesity, insulin resistance and the development of type 2 diabetes mellitus (T2D). Increased obesity, as measured by body mass index (BMI), correlates well with increased serum ob gene protein (leptin, Lep or Ln,), a hormone secreted by adipocytes and involved in the regulation of body fat and alternations in insulin action and secretion. The main objective of this study was to investigate the association between serum free fatty acids and Lep levels in healthy subjects and patients with newly diagnosed type 2 diabetes and their use as potential new targets in the treatment of T2D. The study included 27 healthy subjects and 27 newly diagnosed type 2 diabetics. Standard analytical protocols were used to measure glucose, glycated hemoglobin (HbA1c), lipid profile and BMI. Free fatty acid analysis was performed by tandem gas chromatography-mass spectrometry, while serum Lep levels were determined by ELISA assay. As expected, our results showed significant differences in glucose, HbA1c, Lep, BMI and age of the participants, and levels of lauric, myristoleic, palmitic, stearic, linolenic, arachidic, DGLA, behenic acid, DHA, SFA, MUFA and total FFA in the study groups. In healthy subjects, a negative correlation was found between glucose levels with total cholesterol and Lep, and a positive correlation was observed between Lep and DGLA. In the diabetic group, a significant association was shown between glucose with HbA1c and DTA levels, while Lep was associated with HbA1c levels. Interestingly, significant associations were observed between glucose and HbA1c levels with lipid profile, BMI and age, as well as SFA, PUFA and total FFA in all participants. Our results also showed associations between different types of free fatty acids and the ob gene protein levels in healthy and newly diagnosed diabetic subjects. In conclusion, the obtained results suggest that elevated concentrations of the ob gene protein and various free fatty acids may influence in the development of T2D and, therefore, may be used as potential biomarkers for the onset and early progression of the disease and as possible new therapeutic targets in the treatment of diabetic patients.