In this study, a novel layer-by-layer polyaniline/graphene (PANi/Gr) structure for electrochemical detection of atrazine was developed. Gr film was synthesized by thermal chemical vapor deposition (CVD) method and transferred onto the PANi-predeposited microelectrode. The properties of PANi/Gr film were thoroughly investigated by high-resolution transmission electron microscopy and Raman techniques. The most attractive feature of this system is a suitable microenvironment, which could provide an amplification of the conductive signal, thus may contribute to enhancing electron transfer and subsequently improve the sensitivity in electrochemical measurements. With low detection limit (∼ 43 × 10−12 g/L), acceptable stability and good reproducibility, the proposed electrochemical immunosensor could be advantageously extended for multiplexed detection of other agents of environmental pollution.