Sodium caseinate films were produced using microbial transglutaminase as a protein cross-linking biocatalyst. Basic parameters for the film production, such as buffer type and concentration, pH, temperature, plasticizer concentration and its influence on transglutaminase activity, mold material for film casting, specimen width, and cutting method, were investigated and compared with standardized methods (DIN EN ISO 527-3). Surprisingly, a previously described sodium phosphate buffer (50 mM, pH 8.0) resulted in crystals after drying the films for 48 h. To avoid this deteriorating effect, the buffer system was optimized and finally a Tris-HCl buffer (20 mM, pH 7.0) was chosen for the production of transparent, smooth films without crystallization. Incubation time and temperature during enzyme treatment had a considerable influence on the mechanical properties of the films.