A novel label-free immunosensor based on hyperbranched polyester nanoparticles with nitrite groups (HBPE-NO2), which were synthesized through a simple one-step chemical reaction, was first developed for specific detection of a-fetoprotein (AFP), the tumor marker for liver cancer. The obtained HBPE-NO2 nanoparticles (NPs) were characterized by the proton nuclear magnetic resonance spectroscopy (H-1 NMR), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). And the fabricated process of immunosensor was investigated by attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR), static water contact angles, scanning electron microscope (SEM), cyclic voltammetty (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical performances of the AFP immunosensor were studied. Results indicated the prepared HBPE-NO2-modified immunosensor showed excellent electrochemical properties and satisfactory accuracy for the detection of AFP of the real clinical samples that attributed to the properties of the HBPE-NO2 NPs, which had nanosized structure to increase the specific surface area and unique chemical reactivity for loading capacity of protein molecules. Construction of biosensors using the structure and properties of hyperbranched molecules will offer ideal electrode substrates, which provided more possibilities for the design of biosensor.