Polysaccharides are among the most abundant biopolymers with glycosidic linkages and diverse branches. They have shown many biological activities, including prebiotic and hypoglycemic effects, anti-inflammation, and gut microbiota modulation. In this study, date flesh polysaccharide (DFP) and date seed polysaccharide (DSP) were extracted using the ultrasonic-alkaline extraction method. Chemical structure, functional properties, prebiotic potential, and metabolite formation of Lactobacillus plantarum and Bifidobacterium animalis subsp. lactis with extracted polysaccharides were investigated. Structural analysis of DSP showed substitution of mannose and conformation of galactopyranosyl, mannopyranosyl, and xylopyranosyl, while DFP showed D-glucosyl residues with β configuration with galactopyranosyl and rhamnopiranosyl groups. DSP showed a strong proliferation effect on B. animalis (9.32 ± 0.05 log CFU.g− 1) and L. plantarum (8.66 ± 0.06 log CFU.g− 1). Moreover, B. animalis produced higher short-chain fatty acids when cultured with DSP. Results of a 28-day date fruit (50 gr/day) or date seed (30 gr/day) powder intervention (TRN: IR.UI.REC.1400.023, 2021.07.17) on blood biomarkers revealed that date seed powder could significantly reduce TG, CRP, FBS, and increase RBC and RDW (p < 0.05). Taken together, DSP had a higher prebiotic, hypoglycemic and anti-inflammatory potential compared to DFP. Therefore, date polysaccharides could be further developed as a potential functional product.