Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy (TEM). PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established. Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S. HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection was added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes. The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system, TEM displayed the nanoparticles self-assembled into large network aggregates. It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.