N-glycolylneuraminic acid (Neu5Gc)-containing glycans are a prominent form of aberrant glycosylation found in human tumor cells and have been proposed as cancer biomarkers. The B subunit of the subtilase cytotoxin (SubB) produced by Shiga toxigenic Escherichia coli recognises Neu5Gc containing glycans. We have previously engineered this lectin, SubB2M, for greater specificity and enhanced recognition of Neu5Gc-containing glycans. Here we further explore the utility of SubB2M to detect Neu5Gc tumor biomarkers in sera from patients with ovarian cancer. Using surface plasmon resonance (SPR) we show that SubB2M can detect the established ovarian cancer biomarker, CA125, in a highly sensitive and specific fashion in the context of human serum. These studies established conditions for screening serum samples from patients with ovarian cancer for Neu5Gc glycans. We found that serum from patients with all stages of ovarian cancer had significantly elevated mean levels of Neu5Gc glycans compared to normal controls. Serum from patients with late stage disease (stages IIIC, IV) had uniformly elevated levels of Neu5Gc glycans. Detection of Neu5Gc-glycans using SubB2M has the potential to be used as a diagnostic ovarian cancer biomarker, as well as a tool for monitoring treatment and disease progression in late stage disease.