As an important environmental contaminant, mercury(Ⅱ) has caused worldwide concern. The T-T mismatch based detection for Hg2+ greatly depends on the design of mercury specific oligonucleotide (MSO). This paper optimized the currently reported MSOs by using SYBR Green I. Based on the binding response analysis between several MSOs and Hg2+and further discussion on the secondary structure of sequences, the optimum T-rich sequences was proposed. Hence a fluorescent detection method towards Hg2+ in water samples based on SYBR Green Ⅰ was established. Consequently, the recovery ratios in spiked real water samples ranged from 82.8% to 101.8% with the relative standard derivation of less than 15%. Results show that this method was slightly affected by the environmental matrix, providing accurate detection of mercury ions in real water samples.