Abstract Linoleic acid was isolated effectively from Sambucus williamsii (SW) seed oil which was extracted by high-pressure fluid and its biological activities were investigated. Linoleic acid was isolated from the oil by urea inclusion, the yield was 65.81% and the purity was 92.12%. 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was used to determine the antioxidant activity of linoleic acid. IC₅₀ of DPPH radical scavenging activity of linoleic acid was 61.92 mg/mL. The antiglycemic activity of linoleic acid was evaluated by determining its inhibitory effect on α-glucosidase. The results showed that α-glucosidase was inhibited to a certain extent by linoleic acid (1.56–25 mg/mL). In addition, the hypolipidemic activity of linoleic acid was investigated in vivo using hyperlipidemia mice models fed with the linoleic acid at doses of 1, 2, 4 g/kg BW (body weight). The results showed that serum lipid levels were highly significantly (p<0.01) improved, which indicated the hypolipidemic activity of linoleic acid. The linoleic acid extracted from SW seed oil was proved to possess good antioxidant, antiglycemic and hypolipidemic activity in human diets, which may have industrial use.