Au/poly anthranilic acid/poly vinyl acetate and Au/poly(anthranilic acid-co-3-carboxy-N-(2-thenylidene)aniline)/poly vinyl acetate nanofibers through electrospinning and their modification with covalent tyrosinase (Ty) immobilization was performed. It was realized by surface activation using N-(3-dimetylaminopropyl)-N-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. Electrochemical impedance spectroscopy (EIS), FTIR-ATR, Raman spectroscopy, and bicinchoninic acid assay analyses demonstrated that Ty was stably and covalently bonded onto the nanofibers. Increase in surface roughness [atomic force microscopy (AFM)] and the presence of Cu atoms in the nanofiber composition after enzyme immobilization confirmed the Ty immobilization. The charge transfer resistances of the nanofibers decreased due to changes in the nanofiber surfaces after attachment of enzyme.