High added-value squalene (SQ) was purified in one step from olive oil deodorizer distillates (OODD) using preparative centrifugal partition chromatography (CPC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane-acetonitrile-butanol (1.8:1.4:0.7, v/v/v), leading to the isolation of the target compound in 4 hours, using a preparative 1L column. Furthermore, a fast UHPLC-DAD method was developed and applied for the identification and quantification of SQ in both OODD and purified form. The content of SQ in the initial material was 23.4%, while the purity of the isolated SQ was 95.5%. The recovery of SQ was calculated at 76.3%. The productivity of the process was calculated at 234 mg/h/L.