Micro-Attenuated Total Reflection (ATR) Fourier Transform Infrared spectroscopy was used to investigate sol–gel layers for biosensing applications prior and after glucose oxidase (GOD) immobilization. The changes occurring in sol–gel infrared spectrum after GOD immobilization were clearly evidenced confirming the retaining of the enzyme activity. Moreover, micro-ATR experimental technique allowed us to investigate the spatial distribution of enzyme concentration. The non-destructive nature of our approach also enabled to monitor the time stability of sol–gel layers and of embedded GOD. The temporal evolution of some peaks in infrared spectra of these sol–gel layers was compared with absorption and steady-state fluorescence measurements. The results reported here confirm that micro-ATR infrared spectroscopy can be usefully employed for a non- or minimally invasive detailed characterization of supports for enzyme immobilization.