A new graphene immunosensing platform based on a new electrochemical immunosensor was designed for sensitive screening of carcinoembryoninc antigen (CEA) as a model biomarker in clinical immunoassay. Gold nanoflowers and single-stranded DNA (ssDNA) molecules were initially assembled onto the surface of graphene for the fabrication of the electrochemical immunosensor using layer-by-layer strategy, and then a sandwich-type immunoassay format was developed for CEA detection based on gold nanoflower-labeled signal antibodies. Graphene nanosheets with high conductivity and biocompatibility could greatly enhance the bound force with ssDNA, and improve the analytical properties of the immunosensor. Gold nanoflowers with electrochemical activity provide a large surface area for the label of biomolecules. The graphene platform displayed a good electrochemical behaviour toward the detection of CEA. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.05 to 45 ng mL−1 with a relatively low detection limit of 0.01 ng mL−1 CEA at signal-to-noise ratio of 3. Intra- and interassay coefficients of variation were below 12 %. The feasibility of the electrochemical immunosensor was evaluated for real-life clinical specimens, and no significant differences at the 95 % confidence level were encountered between the immunosensor and commercially available Roche Elecsys 2010 Electrochemiluminescent Automatic Analyzer. In addition, the analytical properties of the immunosensor were comparatively favourable with those of other electrochemical immunosensors.