AbstractCo‐immobilization was applied to combine complementary enzyme reactions. Therefore, trypsin was co‐immobilized together with both, lipase and α‐amylase, onto the surface of non‐woven polyester material. The progress of the immobilization reaction was directly monitored by investigating covalent fixation of the enzymes to the polyester flees using 1H‐MAS‐NMR. Co‐immobilization of the different types of enzymes to the polyester support showed retained enzymatic activity. However, a competition of binding to the support was observed. Increasing amounts of one type of enzyme reduced the degree of immobilization for the other type. In order to investigate the distribution of trypsin and α‐amylase on the polyester support, the flees was treated with a mixture of rhodamine isothiocyanate labeled with anti‐trypsin antibodies and fluorescein isothiocyanate labeled with anti‐α‐amylase antibodies. Using fluorescence microscopy, the co‐immobilization was analyzed by selective excitation of both chromophores at 480 and 530 nm, respectively. In addition, fluorescence spectroscopy was applied by direct labeling of trypsin and lipase prior to co‐immobilization to the polyester support. A special prism of plexiglass was constructed, which fit into a 10 × 10 mm fluorescence cuvette in that way that a diagonal plane was formed within the cuvette. The non‐woven support was fixed in the cuvette and fluorescence spectra were obtained to characterize the amount of different enzymes linked to the support. Using FRET it was demonstrated that a uniform distribution of the various enzyme species was achieved, where the different enzyme activities are bound on the support in close neighborhood to one another. Biotechnol. Bioeng. 2007;96:623–630. © 2006 Wiley Periodicals, Inc.