This study aimed to investigate the association between the serum levels of carbohydrate antigen 199 (CA199), carbohydrate antigen 72 − 4 (CA72-4), carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA) and lymph node metastases in patients with gastric cancer. 200 gastric cancer patients from the First Affiliated Hospital of Hebei North University were selected to determine the concentrations of serum CEA, CA19-9, CA72-4 and CA125. The levels of serum CEA, CA19-9, CA72-4 and CA125 increased with the number of lymph node metastases. The results showed statistically significant differences in the levels of CEA, CA199 and CA72-4 among groups of N0, N1, N2 and N3 (p < 0.05). No significant difference was observed in the CA125 level among the groups of N0, N1 and N2 (p > 0.05), while the level of CA125 in group N3 was significantly higher than that in the other groups (p < 0.05). Comparing to the N0 group, the positive rates of four tumor markers in the N1 + N2 + N3 group were significantly increased in both single-marker detection and combined detection (p < 0.05). In the N1 + N2 + N3 group, the positive rates of combined detection of two, three and four tumor markers were significantly higher than that of the single-marker detection (p < 0.05). The results of combined detection showed no statistically significant difference between the groups that included CA125 and the groups that only contained other tumor markers (p > 0.05). Combined detection of CEA, CA19-9, CA72-4 and CA125 tumor markers may be of great significance in determining lymph node metastases in gastric cancer. CEA, CA19-9 and CA72-4 may have higher clinical value compared to CA125.