The gene encoding a thermostable β-1,3-glucanase was cloned from Thermobifida fusca and expressed constitutively by Yarrowia lipolytica using plasmid pYLSC1. The expression level of the recombinant β-1,3-glucanase reached up to 270 U/mL in the culture medium. After a treatment with endo-β-N-acetyl-glucosaminidase H, the recombinant protein appeared as a single protein band, with a molecular size of approximately 66 kDa on the SDS-polyacrylamide gel. The molecular weight was consistent with the size predicted from the nucleotide sequence. The optimum temperature and pH of the transformant β-1,3-glucanase were 60 °C and pH 8.0, respectively. This β-1,3-glucanase was tolerant to 10% methanol, ethanol, and DMSO, retaining 70% activity. The enzyme markedly hydrolyzed Wolfiporia cocos and Pycnoporus sanguineus glucans. The DPPH and ABTS scavenging potential, reducing power and total phenolic contents of these two Polyporaceae hydrolysates, were significantly increased after 18 h of the enzymatic reaction. The present results indicate that T. fusca β-1,3-glucanase from Y. lipolytica transformant (pYLSC1-13g) hydrolyzes W. cocos and P. sanguineus glucans and improves the antioxidant potential of the hydrolysates.