Prunus amygdalus is used in the folk medicine that proved the interest of this plant which cures many diseases. Many researchers are interested to valorize almond waste (hull and shell) and to evaluate their biological and pharmacological activities. In this work, polysaccharides from Prunus amygdalus shell were extracted sequentially by water, ammonium oxalate and hydrochloric acid. The monosaccharide composition of polysaccharides fractions was performed by GC-MS. Water-soluble polysaccharide was found to be the most effective extracting agent with an extracted yield of 9%. The acid Soluble Polysaccharides (ASP) exhibited the highest galacturonic acid content (31.95%), the highest polysaccharides extractability (88.57%) and the lowest degree of esterification (31.76%). The different polysaccharides fractions were characterized by FTIR, 1H NMR and SEC/MALS/VD/DRI. The antioxidant tests (DPPH, ABTS and FRAP) indicated that ASP showed even better antioxidant activities. Moreover, the result of the antiproliferative activity against Caco-2 and B-16 cells showed that ASP exhibited strong cytotoxicity ability which confirmed that the Prunus amygdalus peels may comprise the natural raw materials for new drug and functional food.