In this paper, we report an alternative simple method to shift the electroactivity of polyaniline (PANI) films to neutral pH conditions by forming multilayer assemblies with poly(anions) using the layer-by-layer (LBL) deposition method. A series of self-assembled PANI multilayer films with poly(anions), such as sulfonated polyaniline (SPANI), poly(acrylic acid) (PAA), poly(vinyl sulfonate) (PVS), and poly(styrene sulfonate) (PSS), were prepared by the LBL method. Their electrochemical behavior and catalytic ability for the oxidation of β-nicotinamide adenine dinucleotide (NADH) in neutral solution were investigated by electrochemistry (EC) combined with surface plasmon spectroscopy (SPS) and the quartz crystal microbalance (QCM) technique. Results indicated that all the films showed very good stability, reversibility, and electroactivity in neutral solution. All the multilayer films can electrocatalyze the oxidation of NADH, with the catalytic ability of PANI/SPANI being higher than that of the other assemblies under the same conditions. The catalytic abilities of the films with the same thickness prepared by the copolymerization method and the LBL method were also compared.