In this work, we prepared large-area, three-dimensional interconnected graphene oxide (GNO) intercalated by self-doped polyaniline nanofibers (SPAN, a copolymer of aniline and m-aminobenzenesulfonic acid) through a simple adsorption and intercalation route via sonication of the mixed dispersions of both components. The strong π–π* stacking between the backbones of SPAN and the GNO basal planes, and the electrostatic repulsion between the negatively charged SPAN and graphene oxide sheets yield a unique free-standing, three-dimensional interconnected nanostructure. The nanocomposite possesses a large specific surface area and maintains a homogenous and stable dispersion with SPAN, which endows it with a high conductivity and good electrocatalytic activity. Because the negative charge and specific structure of the nanocomposite can prompt the adsorption of positively charged guanine and adenine via strong π–π* interactions or electrostatic adsorption, the hybrid was adopted as an excellent sensing platform for highly sensitive determination of guanine and adenine. The electrocatalytic platform exhibited some advantages, such as high sensitivity, good reproducibility and long-term stability.