Comparative optimization of culture media for recombinant XynBTN63D expression in Saccharomyces cerevisiae BJ1824 was performed to identify an efficient and low-cost cultivation medium for sustainable xylooligosaccharide (XOS) production. The gene encoding endo-β-1,4-D-xylanase (XynBTN63D) from Bacillus sp. was cloned into pESC and pYHM1 expression vectors and heterologously expressed in S. cerevisiae BJ1824. Recombinant enzyme production is commonly carried out using Yeast Extract Peptone Galactose (YPG) medium; however, its relatively high cost limits its industrial applicability. Therefore, this study evaluated alternative minimal media to support yeast growth and recombinant XynBTN63D production at a lower cost. Three culture media, namely YPG, MTE, and YMin, were comparatively assessed based on cell growth, protein expression, and enzyme activity. Recombinant protein expression was analyzed by SDS-PAGE, enzyme activity was determined by UV spectrophotometry, and hydrolysis products were characterized by thin-layer chromatography (TLC). Growth analysis demonstrated that the optimal growth of S. cerevisiae BJ1824 was achieved after 72 hrs in YPG and YMin media and after 84 hrs in MTE medium. Among the tested media, YMin showed promising performance, with enzyme activities of 0.930 U/mL and 0.634 U/mL in S. cerevisiae BJ1824 harboring pESC-xynBTN63D and pYHM1-xynBTN63D, respectively. SDS-PAGE analysis revealed a recombinant XynBTN63D protein band at approximately 35 kDa, showing strong expression in YPG medium, weaker expression in Y-Min medium, and no detectable expression in MTE medium. TLC analysis identified xylotriose (X3) as the major hydrolysis product generated by the recombinant enzyme. Overall, these findings demonstrate that YMin medium has strong potential as a cost-effective alternative for recombinant XynBTN63D production in S. cerevisiae BJ1824, thereby supporting more sustainable and economically viable XOS production processes.