A solid phase extraction method based on new functionalized silica gel sorbent for the preconcentration and determination of trace mercury (II) ions by cold vapor atomic absorption spectrometry (CV- AAS) was developed. The effective parameters on mercury extraction were optimized using two optimization methods: fractional factorial design and central composite design. Various factors affecting the recovery of the analyte were investigated using batch equilibrium technique. The best eluent for desorption of mercury was 5 mL of 0.1 M thiourea. Many anions and cations were examined in interference studies and the method shows very high selectivity for determination of mercury in presence of other species. Under the optimum conditions, the analytical curve was found to be linear in the range of 10- 1600 ng L-1 with a detection limit of 2.3 ng L-1. The method was successfully applied to determination of mercury in tap, river, sea water, urine and fish samples with good spike recoveries. The obtained results were in accordance with electrothermal atomic absorption spectrometric method. Student’s t-test indicated the validity and accuracy of the proposed method for analysis of mercury in real samples.