Phasins are proteins found on the surface of natural polyhydroxyalkanoate (PHA) granules. Due to their high affinity for PHA, they can potentially be used as a fusion partner to immobilize other proteins. In this study, we investigated the immobilization of a lipase onto electrospun polyhydroxybutyrate nanofibers. Due to a superior surface area-to-volume ratio, PHB nanofibers retained much larger amounts of enzyme than conventional immobilization supports. More importantly, when used in combination with a phasin tag, the enzyme immobilized on PHB nanofibers exhibited markedly higher activity and reusability. Our approach combines the advantageous features of nanofibrous materials and the regio-specificity of biomolecular interactions for the efficient use of enzymes.