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Metabolomes are small molecule metabolites (<1000 Da) produced by cellular processes. Metabolomes are close counterparts to the genome, transcriptome and proteome. The aim of this study was to develop a method to detect and quantify candidate nucleoside metabolomes 1‐methyl adenosine (1‐MA), 1‐methylguanosine (1‐MG) and 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) in the urine of patients with breast cancer using gas chromatography–mass spectrometry (GC–MS). The method was applied to urine specimens from patients with breast cancer (n = 56) and benign breast tumors (n = 22), as well as from healthy females (n = 20). The relative standard deviations of precision and repeatability analysis were <10%, and recoveries ranged from 88.5 to 105.6%. Limits of detection were 0.014, 0.012, and 0.018 mg/L for 1‐MA, 1‐MG and 8‐OHdG, respectively. The lower limits of quantitation were 0.056, 0.048 and 0.072 mg/L, respectively. There were significant differences in concentrations of candidate metabolomes between patients with cancer and the healthy individuals, especially for those in the early stages of the disease (p < 0.001). No significant differences were observed between the benign and healthy groups. In conclusion, a reliable GC–MS method for the detection and quantification of 1‐MA, 1‐MG, and 8‐OHdG metabolomes in urine has been developed.