Objective To investigate the difference of serum amino acid levels between breast cancer patients and normal volunteers, and explore its significance in breast cancer screening. Methods Totally 59 breast cancer patients (breast cancer group) diagnosed by the Department of Pathology, Chongqing University Cancer Hospital from Apr. to Jun. 2019 were recruited, and 53 normal healthy women were selected as control group. The levels of 20 amino acids in the sera of recruited people were detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). The differences of amino acid levels were analyzed between breast cancer group and control group, between different molecular subtype groups of breast cancer, and between early and advanced breast cancer groups. Receiver operating characteristic (ROC) curve was used to evaluate the effectiveness of different serum amino acids in breast cancer screening. Results The levels of alanine (Ala), asparagine (Asn), citrulline (Cit), glutamic acid (Glu), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), ornithine (Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tyrosine (Tyr) and valine (Val) were higher in the breast cancer group than in the control group (all P<0.05). The level of arginine (Arg) was lower in the breast cancer group than in the control group (P=0.003). ROC curve analysis showed that of the 14 amino acids which were elevated in the sera of breast cancer patients, Ala or Pro alone had better performance in screening breast cancer, and the area under curves (AUCs) were both 0.75; Ile alone had the highest sensitivity, with the AUC of 0.79; Lys alone had the highest specificity, with the AUC of 0.94; and the combined detection of the 14 amino acids improved the performance of breast cancer screening, with the AUC of 0.88, sensitivity of 0.69, and specificity of 0.94. The level of serum Val was higher in early breast cancer patients (TNM stageⅠand Ⅱ) than in advanced patients (TNM stageⅢandⅣ) (P=0.044), and there was no significant difference in the levels of other 19 amino acids (all P>0.05). No significant difference was found in the 20 amino acids between Luminal A type, Luminal B type, triple-negative, and human epidermal growth factor receptor 2 (HER2) positive breast cancer groups (all P>0.05). Conclusion The levels of 15 serum amino acids in breast cancer patients are different from those in normal persons, which is helpful for breast cancer screening. The combination detection of 14 elevated serum amino acids in breast cancer patients can improve the screening performance and may serve as potential biomarkers.