Real-time in situ imaging of organelles is increasingly important in modern biomedical analysis and diseases diagnosis. To realize this goal, organelle-targeting nanoparticles as one of the most commonly used technologies in subcellular sensing and imaging has attracted a lot of interest. The biocompatibility, specificity, and binding efficiency are especially critical for efficient organelle-targeting bioimaging. Gold nanoparticles (AuNPs) fabricated with bifunctional peptides constructed with both Au-binding affinity and nucleus-targeting ability were designed and examined for efficient nucleus-targeting bioimaging. Such a design is expected to achieve an oriented assembling of peptides by the medium of the Au-binding peptides specifically assembled on the surface of AuNPs, with the nucleus-targeting end open for accessibility. The bifunctional peptides showed strong binding affinity toward AuNPs and led to a binding capability ∼1.5 times higher than that of the bare nucleus-targeting peptides, ensuring good surface coverage of the nanoparticles for enhanced nucleus-targeting ability. Such fabricated AuNPs demonstrated over 90% cell viability after incubation for 24 h with HepG2 cells, which were highly biocompatible. Precise and efficient bioimaging of the nucleus was achieved for HepG2 cells by using the fabricated AuNPs as observed with a confocal laser scanning microscope, a dark-field/fluorescence microscope, and a transmission electron microscope. The high surface coverage and oriented binding pattern appeared to be a promising strategy for construction of organelle-targeting agencies.