The conditions were optimised for preparing Alginate oligosaccharide (AOS) nanoliposomes, and Caco-2 cell experiments were carried out to examine their antitumour effects. The optimal formulation of AOS nanoliposomes was as follows: a phosphatidylcholine-to-cholesterol ratio of 5.12, AOS concentration of 8.44 mg/mL, Tween 80 concentration of 1.11%, and organic phase to aqueous phase ratio of 5.25. Under the above conditions, the experimental encapsulation efficiency was 65.84%, and the AOS nanoliposomes exhibited a small particle size of 323 nm. After Caco-2 cells were treated with AOS liposomes and AOS for 24 h, AOS nanoliposomes inhibited the growth of Caco-2 cells to a greater extent than AOS at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL (P < 0.01). LDH leakage exhibited a concentration-dependent increase following treatment with 0.5-1 mg/mL AOS nanoliposomes, and the inhibitory effect of AOS nanoliposomes exhibited a more significant difference than AOS (P < 0.01). Cells treated with 0.5 mg/mL and 1 mg/mL AOS nanoliposomes displayed a substantial and significant increase in activity compared with AOS (P < 0.01). Based on these results, AOS nanoliposomes exerted a more significant effect on inhibiting Caco-2 cell proliferation than AOS.