Buffalo Milk fat has excellent properties; its variable physicochemical properties and its lack of functionality restrict its uses in food industry. The Multi-step dry fractionation was applied on buffalo butter oil (BO) for enhancement of the nutritional value and functionality at three different temperatures i.e. 35, 25 and 15 °C, three solids (S35, S25 and S15) and three liquid (L35, L25and L15) fractions was gained.  LMF (L15and S15) was significantly higher not only in short chain fatty acids (SCFA) but also in long chain unsaturated fatty acids (LCUSFA), whereas, lower in medium saturated chain fatty acids (MCSFA) than those of BO, HMF(S35) and MMF (L25, S25 and L35) were detected. LMF had positive healthy effect of lower oxidative stability index (OSI) and of the highest nutritional (by ω6: ω3 3PUFA) than other fractions. differential scanning calorimeter (DSC) values for both solid and liquid fractions are significantly (P˂0.05) decreased with decreasing fractionation temperature even in melting or crystallization behaviors. Solid fat content (SFC) was significantly the lowest in LMF at 25^OC. The liquid fractions had larger crystals size than those solid fractions and BO. Strong negative correlation between SFC at 20^OC and (USFA, SCFA and IV) and strong positive correlation between SFC and (SFA, MCSFA, LCSFA and DSC) were observed. Results will increase the knowledge to improve the processing condition to produce buffalo butter oil fractions with increased functionality and more varieties for more dairy fat products. The chosen dry fractionation temperature resulted in an increase of the functional and nutritional properties of obtained buffalo butter oil fractions.