In this paper, we report a kind of ordered 3D Au nano-prickle clusters by directly electrodeposited on glassy carbon electrode utilizing the spatial obstruction/direction of the polycarbonate membrane. The proposed 3D nanoclusters are applied to fabricate a sandwich-type electrochemical immunosensor with human IgG as a model analyte. The electrodeposited Au nanoclusters build direct electrical contact and immobilization interface for protein molecules, which do not need post-modification and positioning. Scanning electron microscopy, cyclic voltammetry and alternating current impedance spectroscopy were used to investigate the properties of the modified interface. The deposited Au nanoclusters are stable with good biocompatibility, large specific surface area and high electron exchange capability. Under the optimized experimental conditions, a wide linear range from 1.0 to 10000.0 ng/mL was reached with a detection limit of 0.5 ng/mL. The calibration curve fits a second-order polynomial equation very well (R(2)=0.9914). The developed immunosensor based on Au nano-prickle clusters possesses advantages such as simple fabrication, fast response, low detection limit, wide linear range, easy regeneration, excellent reproducibility and long stability. To our knowledge, the Au nanostructure of special ordered 3D nano-prickle clusters is new for electrochemical immunosensor.