Abstract Human interferon gamma (hIFNγ) is an important inflammatory cytokine, which is extensively expressed by immune system in response to various pathogens. In this work we present a biosensor for the direct detection of hIFNγ based on surface plasmon resonance (SPR) and engineered proteins derived from albumin binding domain (ABD) of protein G. We compare two methods for the immobilization of ABD: covalent coupling and immobilization via streptavidin–biotin interaction. It is shown that both the methods fail to preserve the activity of short ABD binders to hIFNγ due to either low accessibility of the binding site of the scaffold, or disruption of its tertiary structure. We, therefore, employed ABD proteins fused with a helical TolA spacer protein. We demonstrated that concentrations of hIFNγ as low as 0.2 nM can be detected in both buffer and albumin-depleted 2% human plasma using the reported SPR biosensor.