Objectives Vascular endothelial dysfunction leads to the emerging of free oxygen radicals, deficiency of antioxidant system, forming of oxidative stress, inflammatory processes and release of proinflammatory cytokines. These things play big role in the development of primary varicose veins. Prolidase has been reported as an indicator of oxidative stress in diabetes, diabetic neuropathy, non-ulcerous dyspepsia, osteoporosis, polycystic over syndrome and many other diseases. The aim of this study is to evaluate the oxidative stress at venous insufficiency and to provide preliminary knowledge about the role of prolidase enzyme in varicose vein formation. Methods Ninety patients aged between 22 and 80 (47.35 ± 17.69) were included in the study and divided into 3 groups. Group1(n:30)(Serum control group): Patients without venous insufficiency. Group 2(n:30)(Tissue control group(healthy vein group): Patients underwent coronary artery bypass surgery (the remaining portion of great saphenous vein used as coronary artery bypass graft used as normal tissue) . Group 3(n:30)(Varicose vein group): Patients underwent varicose vein surgery (varicose vein and serum of these patients were used for study). Total Oxidant Status (TOS), Total Antioxidant Status (TAS), Oxidative Stress Index (OSI) and Prolidase enzyme levels were detected in tissue and serum samples. Results No significant changes were detected between three groups' serum samples in oxidative stress parameters and in the prolidase enzyme activity. The tissue TOS and OSI were higher in varicose vein group according to normal vein group and this was found statistically significant. And TAC levels in varicose vein group were significantly lower than normal vein group. Prolidase enzyme activity in varicose vein group was found higher according to normal vein group. Conclusion Oxidative stress plays a role at the development of primary varicose veins at biochemical level. Prolidase enzyme related with oxidative stress may play an important role in the pathogenesis of primary varicose veins.