A long-acting formulation of recombinant interferon alpha subtype 2b (IFN-a2b), in which IFN-a2b is coupled, via proline, to polyethylene glycol (PEG), with antiviral, immunomodulating and antineoplastic activities. Upon administration of ropeginterferon alfa-2b, IFN-a2b targets and binds to specific IFN cell-surface receptors. This activates IFN-mediated signal transduction pathways and induces the transcription and translation of genes with IFN-specific response elements (ISREs). Their protein products mediate antiviral, antiproliferative, anticancer, and immune-modulating effects. The PEG moiety inhibits proteolytic breakdown and clearance of IFN-a2b, which prolongs its half-life, extends the duration of its therapeutic effects and allows less frequent dosing. The proline linker facilitates the synthesis of a single positional isomer which further increases its stability and half-life.