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In this study, we designed a fluorescence resonance energy transfer system containing gold nanorods (AuNRs) and fluorescein (FAM) for the detection of hepatitis B virus DNA sequences. AuNRs were synthesized according to the seed-mediated surfactant-directed approach, and the surface of the AuNRs was wrapped with a thin layer of cetyltrimethylammonium bromide (CTAB), resulting in the AuNRs being positively charged. When FAM-tagged single-stranded DNA (FAM-ssDNA) was added into the AuNRs suspension, it was adsorbed onto the surface of the positively charged AuNRs and formed a FAM-ssDNA–CTAB–AuNRs ternary complex, the resulting structure led to a fluorescence resonance energy transfer (FRET) process from FAM to AuNRs and the fluorescence intensity of FAM was consequently quenched. When complementary target DNA was added to the FAM-ssDNA–CTAB–AuNRs complex solution, a further decrease in fluorescence intensity was observed because of an increased FRET efficiency. Under optimal conditions, the decline of the fluorescence intensity of FAM (ΔF) was linear with the concentration of the complementary DNA from 0.045 to 6.0 nmol L−1 and the detection limit was as low as 15 pmol L−1 (signal/noise ratio of 3). When this fluorescent DNA sensor was used to detect the polymerase chain reaction product of hepatitis B virus gene extracted from a positive real sample, a positive response was obtained. Impressively, the biosensor exhibits good selectivity, even for single-mismatched DNA detection. [ABSTRACT FROM AUTHOR]