Introduction: S100B is a neurotrophic factor regulates neuronal growth and plasticity via activating astrocytes and microglia through production of neuro-inflammatory molecules like interleukin (IL)-1β involved in many mental disorders, few studies have combined S100B and cytokines to explore their roles as neuro-inflammatory biomarkers in Generalized Anxiety Disorder (GAD). Methods: Serum S100B and cytokines (IL-1β , IL-2, IL-4 and IL-10) of 108 untreated GAD cases and 123 healthy controls were determined by enzyme linked-immuno-sorbent assay (ELISA) and then compared, while Hamilton Anxiety Rating Scale (HAMA) scores were measured to evaluate anxiety severity. Results: The serum S100B and IL-1β, IL-2 levels of GAD cases were lower than HC significantly (P<0.001), the IL-4 level of GAD were higher than HC (P<0.001), while IL-10 had no significant difference between two groups (P=0.215). The ROC area of S100B, IL-1β, IL-2 and IL-4 in diagnosis of GAD was (0.740 ± 0.032) , (0.900 ± 0.021) , (0.920 ± 0.018) and (0.696 ± 0.037) , all of them suggested a good predicting value (P < 0.001) , while the ROC area of IL-10 was (0.544 ± 0.038) (P = 0.251). The sensitivity of S-100B, IL-1β, IL-2 in diagnosis of GAD was 73.1%, 80.6%, 85.2%, while the specificity was 61.0%, 86.2%, 80.5%. The combination ROC area of S100B, IL-1β , IL-2 and IL-4 was (0.985 ± 0.006)(P < 0.001). Serum S100B was positively correlated with IL-2 and IL-4 (P <0.05)., while was negatively with HAMA scores (P <0.001). Conclusion: The serum S-100B, IL-1β, IL-2 levels of GAD were down-regulated while IL-4 was up-regulated, both IL-2 and IL-4 had a good diagnosis value in GAD separately while the combination of S100B and cytokines had a better diagnosis value which means the neuro-inflammation in GAD is a network regulated by many factors.