Mercury ion (Hg2+) is considered to be one of the most toxic heavy metal ions. Once the content of Hg2+ exceeds the quality standard in drinking water, the living environment and health of human beings will be threatened and destroyed. Therefore, the establishment of simple and efficient methods for Hg2+ ion detection has important practical significance. In this paper, we present a highly sensitive and selective fiber-optic surface plasmon resonance (SPR) Hg2+ ion chemical nanosensor by designing thymine (T)-modified gold nanoparticles (Au NPs/T) as the signal amplification tags. Thymine-1-acetic acid (T-COOH) was covalently coupled to the surface of 2-aminoethanethiol (AET)-modified Au NPs and Au film by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) activation effect, respectively. In the presence of Hg2+ ions, the immobilized thymine combines specifically with Hg2+ ions, and forms an Au/thymine-Hg2+-thymine/Au (Au/T-Hg2+-T/Au) complex structure, leading to a shift in SPR wavelength due to the strong electromagnetic couple between Au NPs and Au film. Under optimal conditions, the proposed sensor was found to be highly sensitive to Hg2+ in the range of 80 nM–20 µM and the limit of detection (LOD) for Hg2+ was as low as 9.98 nM. This fiber-optic SPR sensor afforded excellent selectivity for Hg2+ ions against other heavy metal ions such as Fe3+, Cu2+, Ni2+, Ba2+, K+, Na+, Pb2+, Co2+, and Zn2+. In addition, the proposed sensor was successfully applied to Hg2+ assay in real environmental samples with excellent recovery. Accordingly, considering its simple advantages, this novel strategy provides a potential platform for on-site determination of Hg2+ ions by SPR sensor.