The objective of the current study was to enzymatically characterize immobilized phytase as a glycoprotein on an optimal blend of starch/PAAm nanofibers. Aspergillus niger phytase (phyA) was first expressed in Hansenula polymorpha using formate dehydrogenase promoter and alpha-mating factor signal peptide. Response surface methodology was then used to optimize the different levels of the enzyme, temperature and pH on the immobilized phytase. The electrospun nanofibers showed uniform morphology with an average diameter of approximately 269 nm. Afterward, the kinetic parameters of Km (56 mu M) and Vmax (401 mu mol/min mg) of the immobilized enzyme improved, but the optimum pH and temperature had not changed in comparison with the soluble phytase. The applicability of the nanofiber was confirmed by its outstanding immobilization efficiency (193%) and suitable retention of catalytic activity of the immobilized phytase (50.4% after the tenth repetition). Optimal orientation of the enzyme on the nanofiber using the in silico approach was predicted, which could improve the kinetic properties. The results demonstrate the good potential of H. polymorpha as an efficient expression system for the production of glycoproteins for immobilization.