In this work, we report the production of a layer-by-layer (LbL) film of gold nanoparticles stabilized with carrageenan (carr-AuNPs) interspersed with a conductive polyaniline (PANI) layer. Conventionally, PANI has poor electroactivity in physiological buffers, limiting its using in electrochemical biosensors. The films were prepared on low cost and easy to manufacture flexible gold electrodes (FEAu). Two adsorption sequences were tested for production of the films-PANI/carr-AuNP and carr-AuNP/PANI. The gold nanoparticle size and colloidal stability were characterized. The films were characterized by cyclic voltammetry, UV-visible spectroscopy and atomic force microscopy. The results showed the synergistic effects of the carr-AuNPs (120 nm) and PANI, which improved both the electrochemical response and the stability of the conductive polymer in physiological medium by three times. The presence of the carr-AuNPs in the film caused a significant increase in roughness of the FEAu-modified electrode compared to that of an unmodified electrode, resulting in an increased active electrode area. Studies of film growth by UV-Vis spectroscopy indicated that the deposition mechanisms of both films involved an auto-regulating adsorption process, with the same amount of material adsorbed in each coating step. The PANI/carr-AUNP film showed considerable improvement in stability and conductivity compared to PANI-only films in the physiological environment, which confers advantages for use as a biosensor.