Background: Early detection of cancer provides effective treatment and saves lives. The objective of this study was to determine whether serum gastrokine 1 (GKN1) protein is a gastric cancer specific diagnostic biomarker. Methods: Two hundred normal heathy individuals and 968 patients with gastric, colorectal, liver, lung, breast, pancreatic, ovary, and prostatic cancers were included in this study. We fractionated whole bloods prior to operation. To normalize for sample-to-sample variation, the serum total protein concentration was adjusted to 15 μg/ml with Phosphate Buffered Saline (PBS) and the samples were then incubated at 70oC for 10 min. The serum levels of GKN1 protein were examined using an ELISA kit. The diagnostic value of serum GKN1 protein for gastric cancer detection was evaluated from receiver operating characteristic (ROC) curves. Findings: The serum level of GKN1 protein in healthy individuals (6.62 ± 1.27 ng/ul) was significantly lower in gastric cancer patients (3.42 ± 0.73 ng/ul). At the optimum cut-off (4.8726 ng/ul) for serum GKN1 protein, the sensitivity and specificity was 96% and 97.5% for gastric cancer. Using serum GKN1 protein as the diagnostic reference, the ROC curve showed a satisfactory diagnostic efficacy with an AUC value of 0.9954 (95% CI 0.9919-0.9988). In addition, the diagnostic accuracy at the serum GKN1 protein cut-off was 0.9675. Interestingly, serum GKN1 levels in patients with AGC were lower than those in patients with EGC. The diagnostic accuracies at the optimum serum GKN1 cut-off were 0.952 and 0.9828 for EGC and AGC, respectively. Furthermore, the serum GKN1 levels robustly discriminated gastric cancer patients from the patients with colorectal, liver, lung, breast, pancreatic, ovary, and prostatic cancer with AUC values of more than 0.97. Interpretation: Serum GKN1 protein is a promising specific diagnostic biomarker of gastric cancer. Funding Statement: Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2018R1A2A2A14019713). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: We obtained written informed consent from all participants in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Board (IRB) of The Catholic University of Korea, College of Medicine (MC16SISI0132). No evidence of familial cancer was found in any of the patients.