Nucleosides are indicators of the whole-body turnover of transfer RNA. Based on the activity of cancer cells these molecules could potentially be used as cancer biomarkers, and several studies have determined that the metabolic levels of nucleosides are significantly altered in cancer patients compared to control groups. Here we report a targeted metabolite investigation of serum nucleosides in esophageal adenocarcinoma specimens. We quantified eight nucleosides using high-performance liquid chromatography/triple quadrupole mass spectrometry (HPLC/TQMS) and determined that the metabolic levels of 1-methyladenosine (p <2.14T10-7), N2,N2-dimethylguanosine (p <2.78T10-7), N2-methylguanosine (p <2.48T10-6) and cytidine (p <6.98T10-4) were significantly elevated while the concentration of uridine (p <3.74T10-3) was significantly lowered in serum samples from cancer patients compared to those of control group. Our results suggest that nucleosides could potentially serve as useful biomarkers to identify esophageal adenocarcinoma.