Objective To investigated the N-linked glycans in bladder cancer FFPE tissue sections, and explored abnormal N-linked glycans modification in bladder cancer FFPE tumor tissues sections.Methods An experimental procedure for in situ extraction of N-linked glycans based on FFPE tissue sections was developed. FFPE tissue sections were digested by PNGase F and liberated N-linked glycans. Permethylation was performed to modify the free end of the N-linked glycans. The relative intensity of N-linked glycans was detected by MALDI-TOF/TOF-MS. Database matching was performed to determine possible glycoforms of N-linked glycans. The accuracy of significantly different N-linked glycans as a predictive bladder cancer biomarker were predicted by ROC analysis.Results The data of MALDI-TOF/TOF-MS detection of premethylation-modified N-linked glycans showed that, in tumor and peritumoral FFPE tissue sections of 16 patients with bladder cancer, the relative intensity of N-linked glycans N2H6, N2H7, N2H8, N2H9 (high mannose) and N5H6F1 (complex) were increased significantly in the tumor tissues of bladder cancer. At the same time, N2H5 (high mannose), N3H5(hybrid) and type N3H4, N4H4, N5H6F1S2 (complex) N-linked glycans were significantly decreased. ROC analysis showed that the biantennary N-linked glycan N3H4 (AUC=0.90) and N4H4 (AUC= 0.91) were reliable in distinguishing tumor and peritumoral tissue of bladder cancer patients separately or combined together, and these N-linked glycans may become a potential biomarker for bladder cancer.Conclusion Abnormal N-glycosylation of proteins in bladder cancer FFPE tumor tissue, N-linked glycans N3H4 and N4H4 may be potential biomarkers of bladder cancer.