New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the α2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the α2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic