Deregulation of microRNAs (miRNAs) is implicated in the initiation and progression of gastric cancer (GC). Previous studies have demonstrated that miR-204 was downregulated in GC tissues. However, its expression profile in serum samples and its potential for clinical value remain unknown. Real-time PCR was performed to evaluate the expression level of serum miR-204 in patients with GC. The association between serum miR-204 expression level and the clinical outcome of GC was then investigated. Our results showed that the expression of miR-204 in serum samples from GC patients was significantly lower than that in the healthy controls (P < 0.01). Serum miR-204 expression level of GC patients was significantly upregulated after receiving surgical resection (P < 0.01). In addition, serum miR-204 was associated with lymph node metastasis (P = 0.016), tumor differentiation (P = 0.001), and TNM stage (P = 0.005). GC patients with low serum miR-204 expression had shorter overall survival than those with high serum miR-204 expression (P = 0.004). Multivariate analysis revealed that serum miR-204 expression level was an independent risk factor for this malignant disease (HR = 3.629, 95%CI = 2.828-8.146, P = 0.015). In conclusion, our findings indicate that serum miR-204 may be employed as a novel biomarker for monitoring the treatment response and predicting the prognosis of GC.