N-glycolylneuraminic acid (Neu5Gc) is an aberrant glycosylation found in human tumour cells and has been proposed as a cancer biomarker. We purpose engineered the B subunit of the Shiga toxigenic Escherichia coli (STEC) subtilase cytotoxin (SubB), SubB2M, to make it highly specific for Neu5Gc-containing glycans. This lectin can detect elevated Neu5Gc in serum samples from ovarian cancer patients. In this study we use SubB2M to detect Neu5Gc tumour biomarkers in sera from breast cancer patients. A known human serum glycoprotein, supplemented into normal human serum at a range of concentrations, was detected with SubB2M via surface plasmon resonance (SPR) as our Neu5Gc-containing glycoprotein standard. We then analysed serum samples from normal human females and patients with Stage I - IV breast cancer, plus a cohort of patients with high-risk breast cancer. We also collected serum samples from healthy, cancer-free individuals before and after a 10 – 12 hour fast to assess the impact diet has on serum Neu5Gc levels. Significantly elevated mean levels of Neu5Gc glycans were detected in serum from patients with all stages of breast cancer compared to normal controls. Receiver operating characteristic analyses showed that serum Neu5Gc levels can discriminate breast cancer from normal individuals with high sensitivity and specificity. Analysis of a series of serum samples collected prospectively over time from high-risk breast cancer patients showed a trend for a decrease in Neu5Gc levels immediately following treatment. Preliminary analysis of a healthy, cancer-free cohort showed that all individuals had low levels of serum Neu5Gc compared to breast cancer patients. Analysis of serum Neu5Gc levels pre and post fasting suggested that diet may not be a confounding factor in a Neu5Gc-based diagnostic for breast cancer. Detection of Neu5Gc-glycans using SubB2M may be useful as a diagnostic test for breast cancer and as a tool for monitoring treatment and progression in late stage disease.