Nanofibrous structures are promising for biocatalyst immobilization due to their large surface area which facilitates the enzyme attachment, stability, ease of separation, and fine porous structure. There is limited research available on the change in enzyme activity following interaction with cyclodextrin. In this study, catalase enzyme was immobilized into nanofibrous structures by various techniques, with and without -CD addition, and the enzymatic activity of catalase was evaluated. In addition, catalase--CD complex containing PEO polymer solution was electrospun in between PCL nanofibrous layers as a newly developed technique. The enzyme immobilized nanofibrous structures were characterized by SEM, XRD, and FT-IR analysis methods. Among all the activity tests, best enzyme activity was recorded with catalase--CD physical mixture encapsulated PCL nanofibrous layers. Moreover, the test results indicated that the use of cyclodextrin in immobilization process considerably improves the catalytic activity of the enzyme. (c) 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44404.