Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis worldwide. In this work, a pulse-triggered ultrasensitive electrochemical sensor was fabricated using graphene quantum dots and gold-embedded polyaniline nanowires, prepared via an interfacial polymerization and then self-assembly approach. Introducing an external electrical pulse during the virus accumulation step increases the sensitivity towards HEV due to the expanded surface of the virus particle as well as the antibody-conjugated polyaniline chain length, compared to other conventional electrochemical sensors. The sensor was applied to various HEV genotypes, including G1, G3, G7 and ferret HEV obtained from cell culture supernatant and in a series of fecal specimen samples collected from G7 HEV-infected monkey. The sensitivity is similar to that detected by real-time quantitative reverse transcription-polymerase chain (RT-qPCR). These results suggests that the proposed sensor can pave the way for the development of robust, high-performance sensing methodologies for HEV detection.