Six low molecular weight fenugreek polysaccharides (FP) were isolated and purified by ethanol stepwise precipitation (EFP‐20, EFP‐40, and EFP‐60) and DEAE‐52 cellulose column method (DFP‐0, DFP‐0.15, and DFP‐0.3), respectively. The effects of different separation and purification techniques on the preliminary properties and biological activities of fenugreek polysaccharides were compared. The results showed that the DEAE‐52 cellulose‐eluted fractions had a higher total sugar content and displayed a looser structure. The molecular weights of all six fractions were in the range of 4‐19 kDa, with significant changes in the ratio of galactose to mannose. All six fractions contained α‐D‐galactopyranose and β‐D‐mannopyranose structures. Activity tests showed that all six fractions possessed antioxidant, hypoglycemic and DNA‐protective activities. Among them, the DFP‐0 fraction showed the highest activity. Overall, different isolation and purification methods lead to changes in the properties and bioactivities of FP, which provides a theoretical basis for the development and application of FP in functional foods and drugs.