In the present work, a convenient signal-on electrochemical label-free aptasensor for 17β-estradiol (E2), a typical steroidal hormones endocrine disrupting chemicals, was proposed. 6-mercapto-1-hexanol (MCH) self-assembled monolayer (SAM) modified Au (MCH/Au) electrode was used as the substrate electrode. Graphene is used with bi-functions, not only to adsorb E2 binding aptamer, serving as the recognition element to E2, but also to be assembled onto MCH/Au electrode when sensing E2, to controllably turn on the electron transfer (eT), and further indicate the signal to E2 concentration. With the synergistic effect of DNase I enzyme, highly sensitive detection of E2 was achieved at this aptasensing system, with a linear range from 0.07 to 10 pM and a detection limit of 50 fM. An outstanding selectivity towards E2 was proven for the sensing system by simultaneously detecting 100-fold potential co-existing interferences. The stability and reproducibility were also evaluated to be satisfactory. Spiked real water analysis further indicated its reliability and potential in practical environmental monitoring.