The morbidity and mortality of colorectal cancer (CRC) have been increasing in recent years, and early detection of CRC can improve the survival rate of patients. RNA methylation plays crucial roles in many biological processes and has been implicated in the initiation of various diseases, including cancer. Serum contains a variety of biomolecules and is an important clinical sample for biomarker discovery. In this study, we developed a targeted metabolomics method for the quantitative analysis of nucleosides in human serum samples by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We successfully quantified the concentrations of nucleosides in serum samples from 51 healthy controls, 37 patients with colorectal adenomas, and 55 patients with CRC. The results showed that the concentrations ofN6-methyladenosine (m6A),N1-methyladenosine (m1A), and 3-methyluridine (m3U) were increased in patients with CRC, whereas the concentrations ofN2-methylguanosine (m2G), 2′-O-methyluridine (Um), and 2′-O-methylguanosine (Gm) were decreased in patients with CRC, compared with the healthy controls and patients with colorectal adenomas. Moreover, the levels of 2′-O-methyluridine and 2′-O-methylguanosine were lower in patients with colorectal adenomas than those in healthy controls. Interestingly, the levels of Umand Gmgradually decreased in the following order: healthy controls to colorectal adenoma patients to CRC patients. These results revealed that the aberrations of these nucleosides were tightly correlated to colorectal adenomas and CRC. In addition, the present work will stimulate future investigations about the regulatory roles of these nucleosides in the initiation and development of CRC.