Over the past two decades, birchwood and beechwood xylans have been used as a popular substrate for the characterization of xylanases. Recently, major companies have discontinued their commercial production. Therefore, there is a need to find an alternative to these substrates. Xylan extraction from Acacia sawdust resulted in 23.5% (w/w) yield. The extracted xylan is composed of xylose and glucuronic acid residues in a molar ratio of 6:1 with a molecular mass of ∼70 kDa. The specific optical rotation analysis of extracted xylan displayed that it is composed of the d-form of xylose and glucuronic acid monomeric sugars. The nuclear magnetic resonance analysis of extracted xylan revealed that the xylan backbone is substituted with 4-O-methyl glucuronic acid at the O2 position. Fourier transform infrared analysis confirmed the absence of lignin contamination in the extracted xylan. Xylanase from Clostridium thermocellum displayed the enzyme activity of 1761 U/mg against extracted xylan, and the corresponding activity against beechwood xylan was 1556 U/mg, which confirmed that the extracted xylan could be used as an alternative substrate for the characterization of xylanases.