Human runt-related transcription factor 3 (RUNX3) gene was a new candidate tumor suppressor gene involved in the TGF-β signaling pathway. We evaluated the diagnostic role of RUNX3 gene methylation in serum DNA of esophageal squamous cell carcinoma (ESCC), gastric cancer (GC) and colorectal cancer (CRC) patients. Methylation-specific polymerase chain reaction (MSP) was used to determine the promoter methylation status of RUNX3 gene in serum DNA. The combination of RUNX3 hypermethylation and conventional tumor markers was further analyzed. Hypermethylation of RUNX3 was detected in 51.4% (36/70) of ESCC, 45.2% (28/62) of GC and 41.5% (27/65) of CRC patients, which was significantly higher than that of benign gastrointestinal diseases and healthy donors (p<0.001). Interestingly, aberrant RUNX3 methylation in serum DNA was associated with advanced stage (p=0.027) and lymph metastasis (p=0.032) in ESCC, but not in GC and CRC. Furthermore, the combinational analysis of CEA, CA19-9 and RUNX3 methylation showed a higher sensitivity and no reduced diagnostic specificity than CEA and CA19-9 combination in the three cancers. The serum RUNX3 promoter hypermethylation may be a promising biomarker for the early diagnosis of ESCC, GC and CRC, which was further confirmed by combining with CEA and CA19-9.