Follicle-stimulating hormone (FSH) is an important indicator of ovarian reserve function in women in clinical testing. In this work, a label-free paper-based immunosensor was developed for electrochemical rapid detection of FSH. A hydrophilic channel surrounded with hydrophobic barriers was firstly fabricated on the chromatography paper by wax printing technology. Then three electrodes were screen-printed on the circle zones of the channel, in which one carbon electrode further modified by reduced graphene-oxide /thionine /gold nanoparticles nanocomposites and FSH monoclonal antibody was used as the working electrode to provide sensitivity of the immunosensor. The detection of FSH is based on the decreased electrochemical current of Thi produced from the specific binding of the FSH and anti-FSH, and the decrease of the current is proportional to the concentration of the FSH. The experimental results exhibited that the immunosensor could be used to detect the standard FSH in range of 1-100 mIU/mL with the detection limit of 1 mIU/mL. And the proposed immunosensor had been successfully applied to detect FSH in serum samples.