Objective In order to examine the effect of 4-week combinatorial intervention with exercise training and dietary restriction on serum fatty acids, and to explore the correlation of intervention-induced improvement of serum fatty acid profile with the reduction of body fat and arterial stiffness. Methods Thirty-three obese adolescents were randomized into the intervention (n = 19) and control (n = 14) groups. The participants from the intervention group were subjected to 4-week combinatorial intervention with exercise training and dietary restriction while the participants from the control group maintained regular activities and diet habits. Anthropometry, serum fatty acids and arterial stiffness were measured before and after 4-week intervention. Results The participants upon combinatorial intervention revealed the improved body compositions and serum fatty acid profile, and reduced arterial stiffness when compared with their basal levels and the control participants ( p < 0.05). Moreover, the decrease in myristic acid, stearic acid, arachidic acid, behenic acid, palmitoleic acid, and dihomo-γ-linolenic acid, was associated with the reduction in body fat. A positive correlation between arachidonic acid and left brachial ankle pulse velocity was observed, and the increase in docosahexaenoic acid was associated with the reduction of left brachial ankle pulse wave velocity and the enhancement of right ankle brachial index. Conclusion The 4-week combinatorial intervention is a useful strategy to improve serum fatty acid profile along with the reduction of body fat and arterial stiffness in obese adolescents.