Poly(L-lysine) templated silica and silica/carbon nanofiber nanocomposites are prepared in the presence of acetylcholinesterase from Drosophila melanogaster (Dm. AChE), leading to the formation of a biocompatible electrochemically active nanocomposite structure with high enzyme loading level. Detailed conformational analysis of poly(L-lysine), Dm. AChE and their interactions with the silica and carbon nanofibers is conducted using micro-Raman spectroscopy, while electrochemical impedance spectroscopy is used to probe the rotational mobility of the protein within the poly(L-lysine) templated silica nanocomposites. It is concluded that the enzyme is highly mobile in its active form, while the carbon nanofibers are very efficient electron transfer nanochannels. Based on these results, biosensors with poly(L-lysine) templated silica/nanofiber nanocomposites were developed, utilizing Dm. AChE as the biocatalyst, poly(L-lysine) templated silica nanostructure as the enzyme stabilizing environment and carbon nanofibers as the direct electron transfer channel to the transducer.