In this study, the fluorescence enhancement effect of guanine base on DNA-Cu/AgNCs was reported and fluorescent methods for the determination of Hg2+ or Cu2+ ion were established. The effect of adjacent and inserted guanine base (G) sequences in DNA template on the fluorescence intensity of DNA-Cu/AgNCs was investigated. It was found that G5 sequence extended from the 5′ end of a typical C-riched DNA strand, 5′-CCCTTAATCCCC-3′ (DNA1), demonstrated fluorescence enhancement effect in the preparation of the DNA-Cu/AgNCs. Moreover, this G5-DNA-Cu/AgNCs can be used to determine Hg2+ and Cu2+ ions due to fluorescence quenching by the two metal ions while the DNA1-Cu/AgNCs only can determine Hg2+ ions. Specific assays for Hg2+ ions and Cu2+ ions were established through masking Cu2+ ions by EDTA and Hg2+ ions by NaBH4, respectively. In Hg2+ determination, a linear range from 5.0 nM to 40 nM was obtained with a detection limit of 1.0 nM. In Cu2+ determination, a wide linear range from 0.01 µM to 5.0 µM was obtained with a detection limit of 5.0 nM. The recoveries of 89.6–106.8% for Cu2+ ions and 90.6–106.2% for Hg2+ ions were obtained in the measurements of Hg2+ and Cu2+ ions spiked lake samples, respectively. The results indicate the potential application of the DNA-Cu/AgNCs as a metal ions probes in real samples.