Prostate cancer (PCa) is now the most common male malignant tumor in both Japan and Western countries. Prostate-specific antigen (PSA) has been widely used for the early detection of PCa; however, it is not an ideal biomarker due to its low specificity. Aberrant glycosylation is closely associated with malignant transformation and cancer progression. Recent advances in glycobiology techniques can be applied to the development of novel biomarkers for PCa. We previously identified PCa-associated aberrant glycosylation on PSA, that is, α2,3-linked sialylation as an additional terminal N-glycan on free PSA(S2,3PSA). We then developed a new assay system for the measurement of S2,3PSA utilizing the μTAS method. The area under the curve (AUC) for the detection of PCa with the %S2,3PSA ratio was significantly better than that with total PSA. Another urgent issue in clinical practice for PCa is the over-treatment of patients with a low malignant potential, as aggressive treatment is not necessary. To overcome this problem, it is essential to develop a useful tool for the measurement of the malignant potential. Core2 ,β-1,6-N-acetylglucosaminyltransferase-1 (GCNT1, C2GnT) is a key enzyme that forms core 2-branched 0-glycans. Its expression is associated with the progression of several cancers. We established a mouse IgG monoclonal antibody (mAb) against GCNT1 and examined the relationship of GCNT1 expression with the clinicopathological status of PCa. GCNT1- negative patients were associated with significantly better PSA-free survival compared with GCNT1-positive patients. Furthermore, we established new methods for GCNT1 detection using urine samples of PCa patients. Immunoblotting was used to examine post-digital rectal examination (DRE) urine from PCa patients. Over 90% of GCNT1-positive PCa patients with high concentrations of serum PSA showed extracapsular extension in prostatectomy specimens. In conclusion, the clinical application of glycobiology techniques is a promising approach to develop novel biomarkers for PCa.