Abstract: Squalene is a natural compound with many functional features in inhibiting lipid oxidation, antitumor, and anticancer, preventing cardiovascular disease, and lowing low-density lipoprotein and blood cholesterol. However, the lack of sustainable natural sources and efficient extraction methods limits the application of squalene in food, cosmetic, and pharmaceutical fields. Rice oil deodorizer distillate (RODD) is a by-product of the rice oil refining process, enriching in a lot of squalene. In the present work, a collaborative technology of saponification and column chromatography was employed to isolate and purify squalene from RODD, which not only expands the sustainable natural source of squalene, but also improves the high-value of rice oil processing by-products. The goal of this work was to obtain a quantitative analysis of the separation processes and to assess the feasibility of this method to isolate squalene. Results of studies on selection of optimum condition for extraction by saponification conditions, i.e. temperature, time, solid-liquid ratio and alkali concentration of process and effect of sample pre-treatment on extraction yield were presented. The results indicated that the optimum extraction conditions for the preliminary saponification and enrichment of squalene were saponification temperature 70 ℃, saponification time 60 min, solid-liquid ratio 1:6 g/mL and alkali concentration 2.0 mol/L. Moreover, the extraction rate of squalene was increased with the increasing of extraction dose, which was not conducive to the purity of squalene. Subsequently, hexane was confirmed by thin layer chromatography as an eluent for squalene column chromatography purification. After purification by column chromatography, the yield of squalene was 94% and the purity was 85%. Therefore, squalene in the deodorized distillate of rice oil can be effectively separated from other substances by saponification column chromatography, and the purity is high. Although the collaborative technology of saponification and column chromatography requires large amount of organic solvents that are flammable and environmentally unfriendly, the solvents can be recovered easily and it requires less sophisticated equipment than molecular distillation, and the equipment was operated under atmospheric pressure and lower temperature.