An ultrasensitive immunosensor of Cys/Au@TiO2 based on disposable screen-printed electrodes (SPE) for PIVKA-II detection for hepatocellular carcinoma (HCC) diagnosis was developed by utilizing Cystine (Cys) and nanocomposite Au@TiO2. Firstly, HAuCl4 underwent a reduction reaction with NaBH4, then Au nanoparticles were coated onto TiO2 nanoparticles. Followed, Cys/Au@TiO2 was formed through self-assembly of cysteine to allow the monoclonal antibody of abnormal thrombospondin to bound to the amino group on the surface of the composite by covalent bonding. The mechanism is to determine the changes in the current of the sensor caused by the specific binding of the abnormal prothrombin monoclonal antibody adsorbed by the complex with its antigen. The Cys/Au@TiO2 immunosensor was fully characterized by various analytical approaches and it showed a wide linear testing range of 1–10000 pg mL−1 (R2 = 0.991) and the limit of detection down to 0.77 pg ml−1, with highly sensitivity and specificity. The results showed that the developed immunosensor platform can effectively detect trace amounts of PIVKA-II protein and has potent clinical application for HCC diagnosis. Display omitted •An ultrasensitive immunosensor of Cys/Au@TiO2 for PIVKA-II detection for hepatocellular carcinoma (HCC) diagnosis was developed.•Cys/Au@TiO2 immunosensor has good performance in detecting PIVKA-II.•Cys/Au@TiO2 immunosensor has good stability and selectivity.•Cys/Au@TiO2 immunosensor platform effectively detects trace amounts of PIVKA-II protein and has potent clinical application for HCC diagnosis.