Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35–200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.