Background: Systematic screening for serum-derived biomarkers of cisplatin (DDP) resistance in gastric cancer (GC) remains unreported. The current study aimed to investigate differentially expressed serum miRNAs in GC patients with DDP sensitivity and resistance and their ability to predict cisplatin treatment effectiveness. Methods: The DDP-resistant GC cell line MGC803/DDP was established for screening these dysregulated miRNAs through small RNA sequencing (sRNA-seq), and the miRNA functions were annotated by bioinformatics analysis. RT-qPCR validated the relative miRNA transcription levels in GC cells and sera from 74 GC patients. The associations between the patient clinical parameters and miRNA expression were analyzed. The diagnostic value was evaluated using receiver operating characteristic (ROC) analysis. Results: sRNA-seq identified 35 miRNAs with differential expression between MGC803/DDP cells and MGC803 cells, with miR-3180-3p as the most significantly upregulated miRNA and miR-124-3p as the most significantly downregulated miRNA in MGC803/DDP cells. Clinical data analysis showed that compared with their expression in chemosensitive patients, miR-3180-3p expression was significantly upregulated and miR-124-3p expression was downregulated in chemoresistant GC patients (both p < 0.0001). A combination of the two miRNAs effectively distinguished the chemoresistant GC patients from the chemosensitive GC patients (AUC = 0.946 +/- 0.023, 95% CI = 0.900 - 0.991, p < 0.001, SE = 85.0%, SP = 82.4%). Downregulated miR-124-3p and upregulated miR-3180-3p were significantly correlated with a high TNM stage. Conclusions: The differentially expressed circulatory markers miR-124-3p, miR-3180-3p, and their combination can serve as serum-based biomarkers to predict the therapeutic benefit of DDP in GC.