Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for diseases related to seminal system including hydatid pregnancy, trophoblastic cancer and orchic teratoma. Therefore, in this work, for the first time we have modified a glassy carbon electrode (GCE) with carbon nano onions (CNOs) gold nanoparticles (AuNPs) and polyethylene glycol (PEG) nanocomposite for a highly sensitive and label-free detection of hCG. CNOs/AuNPs/PEG nanocomposite modified GCE shown 150% high electronic conductivity and 150% more surface area. Fabrication of the immunosensor at CNOs/AuNPs/PEG nano composite interface was studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). Under optimum conditions, dose response curve was obtained from 0.1 fg mL(-1) to 1 ng mL(-1) by SWV technique using Fe[CN](6)](3/4-) redox probe. The calibration plot gives a negative linear relationship between log[concentration] of hCG and electrical current with a linear correlation coefficient of 0.99. Experimentally, a detection range 0.1 fg mL(-1) to 1 ng mL(-1) with a limit of detection 0.1 fg mL(-1) was obtained. This immunosensor demonstrated high sensitivity, selectivity, resistant to interference, reproducibility, stability and potential to be used with real samples at healthcare centers. CNOs/AuNPs/PEG nanocomposite interface may also be used to fabricate diagnostics devices for the detection of other biomarkers and protein of clinical significance.