The bladder cancer, which is a kind of a malignant tumor in the bladder mucosa, is the most common malignancy tumor in the urinary system. Diagnosis at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy is closely associated with alterations of glycan expression, but glycosylation status in bladder cancer has not been well studied. Four bladder cancer cell lines (KK47, YTS1, J82, T24) and two normal bladder mucosa cell line (HCV29, HUCV1) were used in our study. We developed a kind of labeling method to derivatize the sialic acid and derivatized the reducing terminal using [¹²C₆]- or [¹³C₆]-aniline, and the derivatized glycans were quantitatively analyzed by Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF/TOF MS). Finally, 52 N-glycans of the bladder cancer were identified. And the sialic acid as well as the fucosylated in the bladder cancer were found significantly over-expressed. At the same time, the bisecting and high-mannose glycan were also high-expressed in the bladder cancer cells. This study provides some referential significance for the future study on the glycomics of bladder cancer.