The performance of porcine interferon-α( pIFN-α) fermentative production by Pichia pastoris is not stable.Cultivation performance in high cell density culture phase is closely associated with the subsequent pIFN-α expression level achieved in induction phase.It was found by analyzing specific activities and transcriptional levels of genes encoding the key enzymes in methanol metabolism that,high( more than 6 g/L) and historic ethanol accumulation in the late glycerol feeding cultivation stage irreversibly deteriorated alcohol oxidase( AOX) promoter,leading to the instability in pIFN-α production.An improved DO-Stat glycerol feeding strategy based on ethanol on-line measurement was thus proposed,to maintain quick cell growth and to control ethanol concentration below 2 g/L simultaneously.Compared with the traditional DO-Stat based glycerol feeding strategy( A),the improved strategy( B) enhanced pIFN-α expression and stabilized fermentation performance,which were showed as follows: 1) under the same induction condition,in two runs,maximal pIFN-α concentration increased 75.5% and 29.8% respectively than that obtained under the best batch using strategy A; 2) reasonably changing induction condition,maximal pIFN-α concentration was still 38.9% higher than that of the best run adopting strategy A.