It is still a challenge to identify mixed polysaccharides isolated from biological tissues. The present study aimed to develop a protocol that allowed for quick characterization of mixed polysaccharides by selecting Cyclina sinensis polysaccharides as test beds. Disaccharides were gradually released by graded acid hydrolysis, separated with 3 kDa molecular weight cut-off membrane, and analyzed by HPLC-ESI-MSn. Furthermore, the monosaccharide composition of the filtrate during the gradient acid hydrolysis process and the final retentate were also determined to provide composition information of these disaccharides. Thus, 6 disaccharides were detected and identified, indicating the existence of polysaccharides constructed of corresponding repeated disaccharide fragments in C. sinensis. The present study demonstrated a potential strategy to characterize mixed polysaccharides without separation and purification.