Separation and purification of secondary metabolites from wild microorganisms remains a challenge due to the analogues existing in the fermentation broth. In this study, a three-liquid-phase salting-out extraction system (TLP-SOES) coupled with column chromatography was developed to purify wortmannilactone F and its dehydrated derivative wortmannilactone L. The TLP-SOES composed of 25.5% petroleum ether/20.0% ethanol/12.9% ammonium sulfate (w/w) at pH 5.6 and room temperature was found to separate wortmannilactone F in the middle (ethanol-rich) phase and wortmannilactone L in the top (petroleum ether-rich) phase with the recovery rates of 88.9% and 83.3%, respectively. The two compounds were further purified by ODS column and semi-preparative HPLC. The total recovery rates of the two compounds were over 2-fold higher than that of the conventional method, which included ethyl acetate/hexane extraction and silica gel column chromatography. Additionally, the calculated logP (ClogP) value of wortmannilactones showed a good linear correlation with their natural logarithm of partition coefficient according to the tie line length (TLL), and that the critical ClogP value might be useful for the selection of TLL or the composition of the extraction system. In summary, our study provided an effective approach for the separation and purification of analogues of natural products.