Osteosarcoma (OS) is the most common malignant bone tumor in patients under 20 years old. Studies have shown that cytokines play important roles in regulating immune responses in OS. In the current study, we investigated the effect of cytokines on OS by assessing serum cytokine profiles. Serum levels of 11 cytokines were measured by multiplex protein arrays in 58 patients with OS and 72 healthy controls. Results showed that serum levels of interleukin 1 receptor antagonist (IL-1Ra), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α) were significantly increased in patients than in controls (2.5-fold, 2.4-fold, 2.7-fold, and 2.1-fold, respectively). When comparing the expression of cytokines in OS patients with different clinical parameters, cases with osteoblastic subtype revealed increased level of IL-6 than patients with other subtypes (p < 0.05); cases with metastasis demonstrated significantly higher level of TNF-α than those without metastasis (p < 0.05), whereas OS patients whose tumor size were bigger than 8 cm presented elevated levels of IL-8 and TNF-α than those with small tumor size (p < 0.05 and p < 0.05, respectively). These data indicated that IL-1Ra, IL-6, IL-8, and TNF-α were associated with increased risk of OS, in which IL-8 and TNF-α may be further correlated with the progression of this disease.