The seeds of tree peony (Paeonia ostii) are promulgated as emerging edible oil crops. However, biological properties of principal constituents of peony seeds were not well studied. Fifteen main constituents including suffruticosols A and B, trans-ε-viniferin, ampelopsin E, resveratrol, trans-resveratrol-4'-O-β-d-glucopyranoside, paeoniflorin, luteolin, luteolin-4'-O-β-d-glucopyranoside, apigenin, kaempferol, oleanic acid, betulinic acid, hederagenin, and caffeic acid were isolated and identified. Their cytotoxicity against human tumor cell lines (COLO205, HT-29, HepG2, AGS, and HL-60) were evaluated. Among them, trans-ε-viniferin showed the most potent cytotoxicity against HL-60 cells (IC₅₀ 5.6 μM); ampelopsin E exhibited the most obvious antiproliferative properties on COLO205 (IC₅₀ 78.1 μM) and HT-29 (IC₅₀ 4.2 μM) cells, and betulinic acid showed the strongest growth inhibitory effects on HepG2 (IC₅₀ 6.6 μM) and AGS (IC₅₀ 5.4 μM) cells. Three enzymes (tyronsinase, α-glucosidase, and acetylcholinesterase) inhibitory activities of 12 compounds were also screened. Stilbene compounds, especially suffruticosols A and B, showed a significant inhibitory activity on all three enzymes. PRACTICAL APPLICATIONS: The cytotoxicity of 15 main constituents from peony seeds against COLO205, HT-29, HepG2, AGS, and HL-60 cells were evaluated. Among them, trans-ε-viniferin showed the most potent cytotoxicity against HL-60 cells (IC50 5.6 μM); ampelopsin E exhibited the most obvious antiproliferative properties on COLO205 (IC50 78.1 μM) and HT-29 (IC50 4.2 μM) cells, and betulinic acid showed the strongest growth inhibitory effects on HepG2 (IC50 6.6 μM) and AGS (IC50 5.4 μM) cells. Collectively, these results suggested that Paeonia ostii seed (POS) extracts are potential candidates for anticancer agents.