Pepsin-colloidal gold conjugates were prepared by a simple protein-friendly process and the enzymatic activity of the bioconjugates is reported. The pepsin-gold conjugates are obtained by mixing colloidal gold and protein solutions at pH = 3 and, thereafter, centrifugation, washing, and redispersion of the pepsin-gold conjugate material in water. The bioconjugates in solution were characterized by W-vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while films of the bioconjugate material obtained by solvent evaporation on suitable substrates were further analyzed by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), transmission electron spectroscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). While TEM and SEM measurements showed aggregates of the enzyme/colloidal gold conjugates, the intactness of secondary and tertiary structures of the enzyme, as determined by FTIR and fluorescence spectroscopies and confirmed by biocatalytic activity measurements, clearly indicates that the enzyme is stable in its natural state and is possibly stabilized by the colloidal gold particles. The enzyme in the pepsin-Au bioconjugate retained substantial biocatalytic activity and was more stable than the free enzyme in solution.