Aim: An accurate and sensitive analytical method was proposed to detect some steroid hormones in biological samples. Materials & methods: An Fe3O4/reduced graphene oxide nanocomposite-based dispersive solid-phase extraction was developed for the effective and simple preconcentration of steroid hormones from human serum samples. Results & conclusion: The nanocomposite was firstly used as adsorbent to simultaneously extract the selected hormones. Limit of detection values for the selected hormones were calculated between 5.5 and 39.2 ng/kg (mass based). An artificial serum sample was used to test the applicability and accuracy of the developed method; percentage recovery results obtained from two different spiked concentrations were found to be in the range of 80.5-99.9%.