Abstract: Objective To observe whether a 548 point C→T single nucleotide mutation of estrogen receptor (ER) affects breast cancer cells (MCF-7) signaling pathway in vitro, and to explore possible mechanism of this ER single nucleotide mutation inducing non-estrogen-dependent precocious puberty.Methods ER gene was inserted into wild-ER pSG5 plasmid as a template using site-directed mutagenesis overlap extension PCR technology to the base 548 points for site-directed mutagenesis. Expression vector of site-directed mutagenesis pSGS-MuER and the ER response element reports luciferase gene vector pGL3-ERE-Luc were constructed. Wild and mutant plasmids were cotransfected with pGL3-ERE-Luc into MCF-7 cells.Luciferase change was observed in order to detect mutation ER reactivity. Results The ER mutants and the ER response element containing the luciferase report gene vector were successfully constructed, pSG5-MuER increased luciferase production than pSG5-ER. Conclusions The mutations of estrogen receptor in vitro lead to high reactivity characteristics. The vector can be used for further reseach.