Estrone(E1), 17β-estradiol(E2) and 17α-ethinylestradiol(EE2) exhibit the highest estrogenic potential. A HPLC-MS-MS analysis method using internal standard is established with the multiple reaction monitoring (MRM) model. Further more, the fragment voltages and collision energies in the MRM model are optimized by response surface method (RSM). The optimal fragment voltages of E1, E2 and EE2 are 205.0, 196.0 and 182.0 V respectively and the collision energies are 43.0, 51.0 and 54.0 V. The established method followed the two-step solid phase extraction (SPE) is then applied for the quantification of E1, E2 and EE2 in the water samples collected from a activated sludge process sewage treatment work and Jialing River at the urban section of Chongqing City. The detection limits of E1, E2 and EE2 are 0.5, 0.4 and 0.1 ng/L.