As a homing peptide, A54 is the most effective peptide specific to the human hepatocellular carcinoma cell. Homing peptide labeled with green fluorescent protein (A54-GFP) was successfully immobilized on the surfaces of magnetic nanoparticles and characterized by Fourier transform infrared spectroscopy as well as fluorescence microscopy. The binding efficiency was analyzed by performing adsorption equilibrium and SDS-PAGE electrophoresis. Specific binding of the nanoparticles functionalized with A54-GFP to human hepatocellular carcinoma cells in vitro was visualized using fluorescence microscopy. The results demonstrated the specificity of A54-GFP-coated magnetic nanoparticle to tumor cell, pointing to its great potential in magnetic cell separation and purification, magnetic resonance imaging (MRI), magnetic hyperthermia, and drug targeting.