Objective To evaluation the value of single and combined detection of peripheral blood human telomerase reverse transcriptase (hTERT) mRNA and tumor markers carcinoembryonic antigen (CEA), carbohydrate antigen 72-4 (CA72-4), CA19-9 and CA125 in the diagnosis of gastric cancer. Methods A total of 48 patients diagnosed with gastric cancer from June 2017 to October 2018 in Baotou Tumor Hospital were enrolled in the study group. Fifty healthy subjects were selected as healthy control group. The peripheral venous blood samples were collected from all subjects. After the synthesis of hTERT cDNA by reverse transcription, real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used for the amplification of hTERT gene fragment. The tumor markers CEA, CA72-4, CA19-9 and CA125 were quantitatively detected by electrochemiluminescence instrument. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of individual markers or joint detection were calculated. The sensitivity and specificity of joint testing items were compared. Results There were statistically significant differences in the positive rates of hTERT mRNA (P< 0.01), CEA (P=0.002), CA72-4 (P=0.003), and CA19-9 (P=0.017) between the gastric cancer group and the healthy control group, while the positive rates of CA125 were not significantly different between the two groups (P> 0.05). The expression of hTERT mRNA was correlated with TMN stage, lymph node metastasis and distant metastasis (all P< 0.05), but was not correlated with age, sex, tumor location, invasion degree and differentiation degree (all P>0.05). The results of diagnostic indices showed that the hTERT mRNA had high sensitivity (84.8％), specificity (82.7％), accuracy (83.7％), positive predictive value (81.2％) and positive likelihood ratio (4.90). CA72-4 also had high specificity (61.2％), accuracy (64.3％), and positive predictive value (45.8％), which were second only to the hTERT mRNA. The diagnostic performance of CA125 combined with CEA, CA72-4, CA19-9 and peripheral blood hTERT mRNA was not significantly different from the latter four combined detection (P>0.05). The sensitivity and specificity of combined detection of traditional tumor markers CEA, CA72-4 and CA19-9 were 62.5％and 80.0％. The sensitivity and specificity of combined detection of hTERT mRNA, CEA, CA72-4 and CA19-9 could be change to 90.0％ and 67.5％. The difference in sensitivity between the two combined detection groups was statistically significant (P= 0.019), and the difference in specificity was not statistically significant (P> 0.05). Conclusion The comprehensive evaluation index of peripheral blood hTERT mRNA is better than the traditional gastric tumor markers, and it is expected to become a new marker for the diagnosis of gastric cancer.