Chemostat culture was performed to characterize the growth, substrate consumption and the hirudin production, and to disclose their interrelations in the fermentation of recombinant Pichia pastoris. The Andrew substrate-inhibited growth model is more suitable than Monod model to simulate the growth of Pichia pastoris on methanol. Therefore, two stationary states can be obtained in the continuous culture at a certain dilution rate because of the substrate inhibition on cell growth. The stationary state could be obtained if only the dilution rate not more than 0.048 h(-1) in the continuous fermentation. The concentrations of cell, methanol and hirudin were constant after 50 h continuous culture with dilution rate at 0.04 h(-1). However, it could not be obtained when the dilution rate more than 0.048 h(-1) because the other stationary point at S > 0.048 h(-1) is unstable. Therefore, it was found that the cell concentration declined and the methanol concentration increased from 2.9 g/L to 18.1 g/L within 18h at dilution rate 0.06 h(-1). Thus, the fed-batch culture with a constant specific growth rate was carried out to disclose the fermentation behavior at high and constant methanol concentration in aid of a methanol sensor. The theoretical maximum specific growth rate, microm = 0.0464 h(-1), was found under critical methanol concentration, Scrit = 3.1 g/L. The growth of P. pastoris was typically methanol-limited at the methanol concentration S < Scrit. It was, however, inhibited at S > Scrit. The maximum specific Hir65 production rate qp was obtained at 0.2 mg/(g x h) when methanol concentration and mu were 0.5 g/L and 0.02 h(-1), respectively. The specific Hir65 production rate qp increased with the increase of mu and S at mu < 0.02 h(-1), and decreased at mu > 0.02 h(-1). The specific methanol consumption rate increased with the increase of S when S < 5 g/L, but decreased when S > 5 g/L. At last, the high Hir65 production rate 0.2 mg/(g x h) was obtained in the fermentation conducted under methanol-limited concentration and mu controlled at 0.5 g/L and 0.02 h(-1), respectively, while the specific methanol consumption rate is low only at 0.04 g/(g x h), showing the potential for the strategy of getting high Hir65 production rate at the low consumption of methanol.