Human interferon alpha 2b (IFN alpha 2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN alpha 2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN alpha 2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN alpha 2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN alpha 2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN alpha 2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN alpha 2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN alpha 2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN alpha 2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN alpha 2b compared to standard IFN alpha 2b produced by E. coli.