A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.