Enzymes cleaving the biopolymer adhesives of fouling organisms are attracting attention for the prevention of biofouling. We report a versatile and robust method to confine the serine protease Subtilisin A (or Subtilisin Carlsberg) to surfaces to be protected against biofouling. The approach consists of the covalent immobilization of the protease onto maleic anhydride copolymer thin film coatings. High-swelling poly(ethylene-alt-maleic anhydride) (PEMA) copolymer layers permitted significantly higher enzyme loadings and activities than compact poly(octadecene-alt-maleic anhydride) (POMA) films. Substantial fractions of the immobilized, active enzyme layers were found to be conserved upon storage in deionized water for several hours. Ongoing studies explore the potentialities of the developed bioactive coatings to reduce the adhesion of various fouling organisms.