A novel electrochemical immunosensor for sensitive detection of cancer biomarker alpha-fetoprotein (AFP) is described that uses a graphene sheet sensor platform and functionalized carbon nanospheres (CNSs) labeled with horseradish peroxidase-secondary antibodies (HRP-Ab2). Greatly enhanced sensitivity for the cancer biomarker is based on a dual signal amplification strategy: first, the synthesized CNSs yielded a homogeneous and narrow size distribution, which allowed several binding events of HRP-Ab2 on each nanosphere. Enhanced sensitivity was achieved by introducing the multibioconjugates of HRP-Ab2-CNSs onto the electrode surface through "sandwich" immunoreactions. Second, functionalized graphene sheets used for the biosensor platform increased the surface area to capture a large amount of primary antibodies (Ab1), thus amplifying the detection response. On the basis of the dual signal amplification strategy of graphene sheets and the multienzyme labeling, the developed immunosensor showed a 7-fold increase in detection signal compared to the immunosensor without graphene modification and CNSs labeling. The proposed method could respond to 0.02 ng mL(-1) AFP with a linear calibration range from 0.05 to 6 ng mL(-1). This amplification strategy is a promising platform for clinical screening of cancer biomarkers and point-of-care diagnostics.