Abstract As(III) is a highly toxic and predominant arsenic species especially in anoxic groundwater. In this paper, the measurement of trace amount of As(III) was reported by using the Au-PANI-Fe-CNFs composite as sensing platform. The Au-PANI-Fe-CNFs composite was constructed by the formation of polyaniline (PANI) nanosheet array on the electrospun Fe-containing carbon nanofibers (Fe-CNFs) substrate followed by self-deposition of Au nanoparticle. PANI presents a uniform array structure on the fiber surface. Au nanoparticles were deposited uniformly on the PANI nanosheet surface by means of the reducing property of PANI, with the particle size of about 20 ± 6 nm. The presence of Fe in the CNFs accelerates the growth of PANI nanosheet and improves the As adsorption in the sensing process of As(III) thereafter. The obtained Au-PANI-Fe-CNFs exhibit excellent electrochemical performance for the detection of As(III) in water, because of the unique structure of PANI array and uniform distribution of AuNPs. The designed electrode displays wide linear range (5– 400 ppb) and high sensitivity with a detection limit of 0.5 ppb (S/N ≥ 3). This novel modified electrode shows a great opportunity for the electrochemical analysis of arsenic and its analogues in water.