A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae alpha-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZ alpha A-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040 x 10(6) IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source.