Immobilization procedures are a fundamental step in the technological use of enzymes. Among the different immobilization procedures sol–gel technique is widely recognised as a valuable approach to obtain very high quality catalytic supports. In this paper different optical techniques have been used and compared to investigate structural and dynamic properties of glucose oxidase (GOD) prior and after sol–gel immobilization process. In particular, using Fourier Transform infrared micro-spectroscopy and time-resolved fluorescence the secondary structure of GOD and the flavin adenine dinucleotide (FAD) conformational changes have been respectively investigated. Infrared spectroscopy measurements have confirmed that enzymatic activity is preserved and a predominant β-sheet subcomponent is retained by immobilized GOD. By time-resolved FAD fluorescence a three-exponential decaying behaviour has been observed for both free and immobilized enzymes with three different lifetimes, each being characteristic of a peculiar conformational state of the FAD structure. The comparison between lifetime values for free and immobilized GOD has not shown significant differences, while the fractional steady-state intensities of the single exponential components have been changed by immobilization procedure. All results reported and discussed in this paper have confirmed once again the efficacy of the adopted sol–gel immobilization procedure for enzymes and proteins. In addition, the joint use of different optical spectroscopic techniques has shown to be a very valuable tool for getting a better insight into structural and dynamic properties of immobilized enzymes.