Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (P-AOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters P-GAP, P-GAP(m), and P-TEF1 and signal peptides alpha-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 x 10(5) U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 x 10(5) U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 x 10(5) U/mL. Finally, LHyal activity of 2.12 x 10(6) U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 x 10(4) U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.