Abstract: Objective To select a suitable housekeeping gene to be the internal reference gene for the investigation of gastric cancer tissues. Methods Fifty pairs of gastric cancer tissues and corresponding adjacent tissues were obtained from the patients with gastric carcinoma undergoing tylectomy. Total RNA was extracted using Trizol from the above tissues, gastric cancer cell lines and normal gastric epithelial immortalized cells. Absolute qRT-PCR was employed to detect the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin(ACTB), RNA polymerase subunit Ⅱ (RPⅡ) and 18sRNA in the gastric cancer tissues and cells. Relative qRT-PCR was used to detect the expression level of carcinoembryonic antigen(CEA) in gastric cancer cells and tissues with the GAPDH, 18sRNA, ACTB and RPⅡ as the housekeeping gene respectively. geNorm software was used to screen the most suitable reference gene for qRT-PCR. Results Comparing with corresponding adjacent tissues, GAPDH, ACTB and RPⅡ expression were obviously up-regulated by (6.49±1.12), (5.02±0.87) and (3.68±0.78) fold in cancer tissues (P<0.05). Yet 18sRNA had no obvious expression change in gastric cancer and corresponding adjacent tissues (P>0.05). The expression of GAPDH, ACTB, RPII and 18sRNA all showed no obvious changes in gastric cancer cell lines compared with normal gastric epithelial cells. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated by (2.87±0.56) fold in gastric cancer tissues. Conclusion There is no universal reference gene in various tissues and cells. 18sRNA is stably expressed in gastric samples and could be used as the most optimization of Housekeeping Gene.