Polyaniline (PANI) nanofibers are synthesized by dilute polymerization and subsequently used for layer-by-layer assembly with poly(acrylic acid) (PAA). The chemical synthesized PANI nanofibers are characterized by SEM and TEM. In addition, the LBL assembly process is characterized by SEM, UV–vis spectrometry and electrochemical methods. PAA inside the multilayer film can dope nanostructured PANI effectively and shift its electroactivity to a neutral pH environment. Compared with PANI/PAA film co-deposited on the electrode by electropolymerization, the redox behavior of PANI/PAA multilayer via LBL assembly is more reversible, indicating the enhancement of electron transfer. The obtained nanostructured PANI/PAA multilayer films are very stable and show high electrocatalytic ability toward H2O2, which makes it an ideal substrate for H2O2 detection and offers great promise for biosensing.