Epithelial ovarian cancer (EOC) is a common malignancy, usually diagnosed at an advanced stage, resulting in over 4,000 deaths each year in the UK. There is currently no National Screening Programme and no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We have studied the biology and function of EN2, and have investigated the potential role as a biomarker in EOC tissue and bodily fluids. Elevated En2 mRNA expression levels and protein staining were observed in EOC tissue, but levels were much lower in benign tumours, and negligible in normal ovary. Therefore EN2 could be utilised as a diagnostic biomarker, particularly in high-grade serous tumours (HGSOC), however this would require an invasive biopsy specimen. Urine samples are more easily obtainable and EN2 protein was positive in 86% of newly diagnosed HGSOC, compared with female healthy controls, making it a potential non-invasive diagnostic biomarker and possible screening tool. In HGSOC patients who received neoadjuvant chemotherapy prior to surgery, elevated En2 mRNA levels and positive protein expression predicted a shorter progression-free survival and resistance to platinum chemotherapy. Such information could be used as a prognostic biomarker to guide post-operative treatment decisions. In normal adult Purkinje neurons EN2 is located in the nucleus at 33kDa, however EN2 was cytoplasmic and visualised at 43kDa or 50kDa in EOC, suggesting post-translational modification from the native state. En2 over-expression studies, including microarray analysis and cisplatin-challenge, suggested a role in the development of platinum-resistance. EN2 was also implicated in cell invasion and metastasis, via the process of epithelial-mesenchymal transition (EMT). EN2 appears to be actively involved in disease progression and treatment resistance in EOC, and further research determining its role in EMT, and the TGFβ receptor and Wnt signalling pathways is warranted. It also shows potential as a clinical biomarker, especially in urine where the non-invasive nature of the test and the potential for detection prior to any clinical signs or symptoms, make it a worthy candidate for ongoing research.