The protein hemoglobin (Hb) or myoglobin (Mb) in a variety of net surface charge states at different pH values was alternately assembled with negatively charged polysaccharide dextran sulfate (Dex) into layer-by-layer (LBL) films on solid surfaces. The proteins in the LBL {Dex/protein}(n) films demonstrated a pair of well-defined cyclic voltammetric (CV) peaks at about -0.35 V vs SCE at pH 7.0 for the heme Fe(III)/Fe(II) redox couple at pyrolytic graphite electrodes, and a sensitive Soret spectroscopic absorption band at around 410 nm on quartz slides. Quartz crystal microbalance (QCM), CV, and UV-vis spectroscopy were then used to characterize the {Dex/protein}(n) films. The interaction between the proteins and Dex under different conditions was investigated, and the driving forces for the LBL {Dex/protein}(n) film assembly were explored. The factors influencing the protein adsorption on the Dex layer surface, such as the concentration of proteins, the ionic strength of protein solutions, and the applied electric field, were also studied in detail. The results suggest that electrostatic interaction is the most important driving force for the film assembly. Even when the proteins had net negative charges on their surface the localized Coulombic attraction between the positively charged surface groups of the proteins and the negatively charged Dex surface would lead to a stable film assembly, although the amount of adsorbed proteins in this case was less than that when the proteins had net positive charges on their surface.