Living organism’s defensive systems are very sensitive and complex mechanisms. Endogenous production of reactive oxygen species (ROS) is one of the main way these systems can achieve their goals. On the other side, ROS can be dangerous also for the living cells and the right balance between pro-oxidant and antioxidant is essential to avoid oxidative stress. Peripartum is typically a very stressful period of life in producing animals and dairy cows can experience a negative redox status at this time. The aim of the trial was to study the relationship between negative energy balance markers such as NEFA and BHBA and oxidative stress in transition dairy cows. Thirty Holstein Friesian dairy cows over 2 commercial farms located in Milan area were involved in the trial. Blood samples were collected at dry-off, at calving and at 30 DIM. ROS and serum antioxidant capacity (SAC) were measured on samples and the Oxidative Stress index (OSi) was calculated. On the same samples NEFA, BHBA and α-tocopherol concentrations were determined. Statistical analysis was performed with MIXED and CORR procedures of SAS. NEFA and BHBA increased around parturition compared with dry off levels (P < 0.05). At 30 DIM, NEFA decreased to concentrations similar to pre-partum, BHBA concentration instead remained high. α-tocopherol blood levels decreased at calving and increased at 30 DIM at higher values than dry-off (3.12 vs.. 3.59 μg/mL, P < 0.05). ROS results were not significant over the time and SAC values showed a tendency (P = 0.05) to have lower concentration at calving compared with dry-off. OSi results were significantly higher at calving compared with pre-calving (0.167 vs.. 0.214, P < 0.05) and 30 DIM indexes (0.214 vs.. 0.178, P < 0.05). There was a positive correlation between NEFA (0.44, P < 0.05) and BHBA (0.28, P < 0.05) serum levels and OSi. The results observed, consolidate the goodness of OSi in detecting oxidative stress in transition cows.