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Cytokeratin-19 (CK19) has been proven to be commonly expressed by cancer cells in a variety of solid tumors and may serve as a suitable marker of metastases in gastric cancer (GC). Since objective assessment of peritoneal lavage or fluid for free cancer cells (FCC) is essential for clinical decision making in patients with GC, it is important to develop a quantitative and reproducible method for such evaluation. We assessed the possible application of One-Step Nucleic Acid amplification (OSNA) assay as a rapid method for FCC detection in intraoperative peritoneal lavage or fluid of GC patients. Seventy-eight intraoperative peritoneal lavage or fluid samples were eligible for the analysis by conventional cytology and OSNA examination. The concentration of CK19 mRNA in intraoperative peritoneal lavage and fluid was compared with the conventional cytological assessment. CK19 mRNA concentration was detected by OSNA assay. For peritoneal lavage samples, sensitivity and specificity were 83.3% and 87.8%, respectively. In peritoneal fluid, significantly higher CK19 values were observed in patients with serosal infiltration (medians: 100 copies/µL vs. 415.7 copies/µL; p = 0.0335) and lymph node metastases (medians: 2.48 copies/µL vs. 334.8 copies/µL). OSNA assay turns out to be an objective, fast, and reproducible quantitative method of FCC assessment.