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ZnS nanoparticles were prepared by hydrothermal method and modified with mercaptoacetic acid in this paper. The functionalized nanoparticles were characterized using transmission electron microscopy (TEM), X-ray diffractometer (XRD) and photoluminescence spectroscopy. They were used as fluorescence probes in the determination of uracil and thymine. Under the optimum conditions, the fluorescence of functionalized ZnS nanoparticles was quenched by uracil and thymine, respectively. The responses are linearly proportional to the concentrations of uracil and thymine both between 0.8 × 10−5 and 9.6 × 10−5 mol L−1and the limits of detection are 0.9 × 10−6 and 0.4 × 10−5 mol L−1 for uracil and thymine, respectively.