Nuclear matrix protein 22 (NMP22) is a crucial bladder cancer marker, the accurate detection of which is conductive to improving the effectiveness of tumor prevention and treatment. However, due to low sensitivity, it is impossible for conventional immunoassay to detect the trace of NMP22 at the early stages of tumor progression. It is crucial to develop a simple, sensitive and accurate detection method of NMP22. As a variety of two-dimensional (2D) material, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) demonstrates excellent conductivity and contains abundant amino groups. As a sort of 3D material, silica-modified Cu-based metalorganic frameworks deposited silver nanoparticles (Cu-MOFs@SiO2@AgNPs) composites are characterized by large surface area, excellent biocompatibility and water stability, which allows the immobilization of anti-NMP22 in large amounts via Ag-N bonds. Through a combination of the advantages exhibited by 2D material, 3D material, AgNPs and disposable screen-printed electrode, a disposable and sensitive label-free electrochemical immunosensor has been successfully assembled for NMP22 detection. In addition to exhibiting a wide linear range (0.1 pg∙mL−1-1000 ng∙mL−1) and a low detection limit (33.33 fg∙mL−1, S/N = 3), the proposed immunosensor also demonstrated satisfactory selectivity, strong reproducibility and high stability. Moreover, the excellent performance in detecting human urine samples makes it applicable in practice.