Adjuvant chemotherapy is an important component in the treatment of gastric cancer (GC) patients; however, some patients do not respond to the drugs. We aimed to develop a practical profile based on serum microRNAs (miRNAs) that can be used to predict patients likely to respond to treatment. Microarrays were used to screen cisplatin-resistant SGC7901/DDP GC cells and the parental SGC7901 cell lines for miRNAs related to chemotherapy sensitivity. The correlation between the expression patterns of identified serum miRNAs and overall survival was confirmed in 68 GC patients. Furthermore, we also validated the signature of the serum miRNAs in an independent cohort of 50 GC patients. From the screening microarrays, we focused on miR-15a, miR-15b and miR-93 as downregulated miRNAs in the SGC7901/DDP cells and miR-27a, miR-106a and miR-664 as upregulated miRNAs. Only serum miR-106, miR-15a, miR-93 and miR-664 were useful in predicting the prognosis of patients who received adjuvant chemotherapy. We identified a signature of four serum miRNAs (miR-106, miR-15a, miR-93 and miR-664) that, when combined, can be used as a risk score for overall survival. Patients with a higher risk score had worse prognosis (p < 0.05). For the independent cohort of patients, the signature of the four miRNAs predicted prognosis well. Our data showed that the risk score derived from the four serum miRNAs was closely associated with the overall survival in GC patients who received adjuvant chemotherapy.