The influence of a catalase (Cat) layer located at different depths in the layer-by-layer hemoglobin/ polystyrene sulfonate films with an (Hb/PSS)(20-x)/(Cat/PSS)/(Hb/PSS)(x) (x = 0- 20) architecture on kinetics of hemoglobin degradation under treatment with hydrogen peroxide solutions of different concentrations and features of H2O2 decay in surrounding solutions has been studied. While assembled on the top of the multilayers, the catalase layer shows the highest activity in hydrogen peroxide decomposition. Hemoglobin in such films retains its nativity for a longer period of time. The effect of catalase layers is compared with that of protamine, horseradish peroxidase, and inactivated catalase. Positioning an active layer with catalytic properties as an outer layer is the best protection strategy for layer-by-layer assembled films in aggressive media.