A simple, rapid and sensitive method for simultaneous determination of six steroid sex hormones in urine matrix was developed by the combination of stir bar sorptive extraction (SBSE) with high performance liquid chromatography (HPLC) and diode array detection (DAD). A poly (methacrylic acid stearyl ester-ethylene dimethacrylate) was synthesized and selected as SBSE extraction medium. To achieve the optimum extraction performance, several parameters, including agitation speed, desorption solvent, extraction and desorption time, pH value, inorganic salt and organic solvent content of the sample matrix were investigated. Under the optimized experimental conditions, low detection limits (S/N=3) and quantification limits (S/N=10) of the proposed method for the target compounds were achieved within the range of 0.062-0.38 and 0.20-1.20 ng/mL, respectively from spiked urine, respectively. The calibration curves of six steroid sex hormones showed good linearity ranging from 1.0 to 200 ng/mL with linear coefficient R2 values above 0.990. Good method reproducibilities presented as intra- and inter-day precisions were also found with the R.S.D.s less than 9.2 and 10.0%, respectively. Finally, the proposed method was successfully applied to the determination of the target compounds in a urine sample from a pregnant woman.