Currently processes of ethanol production from desulfurated red seaweed derived polysaccharides (De-RSDP) are well established. However, the optimization of the enzymatic saccharification process has not been reported. In this study, De-RSDP from Kappaphycus alvarezii was subjected to saccharification by different enzymes, including pectinase, cellobiase, cellulase, and hemicellulase. The best saccharification enzyme was determined as pectinase. In order to obtain more reducing sugar (RS), the single-factor experiment followed by central composite rotatable design (CCD) was used to optimize the parameters for enzymatic hydrolysis. The optimal saccharification parameters of De-RSDP were 4400 U/g pectinase dosage, 96 hr, and 55.5°C, respectively, and the yield of RS reached 81.3%. Subsequently, the saccharification liquids of De-RSDP were isolated and purified with gel permeation chromatography (GPC) to separate saccharides with different molecular weights. Simultaneously, the composition of saccharification liquids was analyzed by 1-phenyl-3-methyl-5-pyrazolone high-pressure liquid chromatography (PMP-HPLC) and mass spectroscopy (MS). The result showed that the content is mainly composed of galactose (65.2%) with the average molecular mass in the fourth peak 181.1 Da. The saccharification liquids of De-RSDP inoculated with Saccharomyces cerevisiae L-4 for ethanol fermentation produced 0.3 g ethanol/g De-RSDP, which corresponded to 71.8% of the theoretical yield (0.38 g ethanol/g).