Various plants and plant products (e.g. vegetable oil) are the major sources of phytosterols. Phytosterols are naturally occurring in either free form, or esters of fatty acids or glycoside forms, depending on their natural sources. As a potential industrial product, the main source is from deodoriser distillate (DOD) which is a by-product of oil refinery. Major applications of phystosterols are used as a functional food additive and a building block. Phytosterols are found to have cholesterol lowering effect by the inhibition of cholesterol absorption in the intestine lumen. As a building block, phytosteryl derivatives can be developed and introduced to pharmaceutical and cosmetic industries. Recently, we have successfully converted phytosteryl ester from industrial rapeseed and soybean oil DOD ethanolysed mixture to free phytosterols by using enzymatic transesterification. This study attempted to optimise the proper method for isolation of free phytosterol from the reaction mixture with the high yield and purity. Also, some characteristics of phytosterol isolated from the reaction mixture were examined. The extraction and partial characterisation of phytosterol from mixed rapeseed and soybean oil DOD were investigated following the ethanolysis reaction catalysed by Novozyme 435. Ethanolysis converted phytosteryl esters to free phytosterols and fatty acid ethyl ester (FAEE). The FAEE was removed by vacuum distillation and the residual ethanolysed products were subjected to isolation of free phytosterols using five washing-crystallization systems including; (A) washing with hexane 2 times and crystallisation with hexane, (B) washing with acetone 2 times and crystallisation with acetone, (C) washing with ethanol 2 times and crystallization with ethanol, (D) washing with hexane, acetone:ethanol (3:2,v/v) and crystallisation with acetone:ethanol (3:2,v/v) and (E) washing with hexane, acetone:ethanol (4:1,v/v) and crystallisation with acetone:ethanol (4:1,v/v). The yield and purity of phytosterol extracts were analysed by HPLC and TLC. Thermal properties and IR spectra were revealed by DSC and FTIR. From the results, system A recovered the highest yield (47.3%) of phytosterol extracts with 83.3% purity. The highest purity (97.3%) with a yield of 30.7% was found in the extracts of system E. All extracts exhibited the same Rf as cholesterol in TLC plate suggesting the predominant of phytosterols. The dominant spectral features in the same region of 1,000-1,500 cm-1 and 2,850-2,395 cm-1 with the different peak height were noticeable for all phytosterol extracts. The intense bands at 1,049 cm−1 corresponding to the secondary C-OH and OH group at 3,602 and 3,679 cm−1 were found. Additionally, the presence of the bands above 3,000 cm-1 indicated the free form of phytosterols. The melting point of all extracts was found to be 130-141ºC. Phytosterols crystallised by system E exhibited the sharp melting peak between 138.5-140.6 ºC. As a consequence, washing with hexane prior to crystallisation with acetone:methanol (4:1, v/v) was an effective method for recovery of free phytosterols from DOD ethanolysed products.