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Background Oriented immobilization of enzymes provides substantial advantages in terms of improving the efficiency of enzymatic process as well as enhancing the reusability of enzymes. Phasins have high binding affinity to PHA, and thus can potentially be used as a fusion partner to immobilize enzymes onto PHA supports. However, presently available PHA supports of granular configuration have low surface-area-to-volume ratio, which limits enzyme loading on the supports. In this study, we explored the use of electrospun PHB nanofibers as an alternative support for high density immobilization of a phasin-fused lipase in directed orientations.Results The electrospun PHB nanofibers were successfully used for selective immobilization of the phasin-fused enzyme, and could anchor 120-fold more enzyme than PHB granules of the same weight. The enzyme immobilized onto the PHB nanofibers exhibited exceptionally high stability. Furthermore, via oriented immobilization of the enzymes, PHB nanofibers loaded with the phasin-fused lipase provided 3- to 10-fold higher enzyme activity than the conventional immobilization supports.Conclusions Our approach combines the advantageous features of nanofibrous materials and the regio-specificity of biomolecular interactions for the efficient immobilization of enzymes, which can be widely adopted in the development of various enzymatic processes.