Over the past years, significant efforts have been devoted to explore novel drug delivery and detection strategies for simultaneous therapy and diagnostics. The development of biotinylated polyurethane-urea nanoparticles as theranostic nanocarriers for targeted drug and plasmid delivery, for fluorescence detection of human hepatocellular carcinoma cells, is described herein. These targeted nanoparticles are specifically designed to incorporate biotin into the polymeric matrix, since many tumor types overexpress receptors for biotin as a mechanism to boost uncontrolled cell growth. The obtained nanoparticles were spherical, exhibited an average diameter ranging 110–145 nm, and showed no cytotoxicity in healthy endothelial cells. Biotinylated nanoparticles are selectively incorporated into the perinuclear and nuclear area of the human hepatocellular carcinoma cell line, HepG2, in division, but not into growing, healthy, human endothelial cells. Indeed, the simultaneous incorporation of the anticancer drugs, phenoxodiol or sunitinib, together with plasmid DNA encoding green fluorescent protein, into these nanoparticles allows a targeted pharmacological antitumor effect and furthermore, selective transfection of a reporter gene, to detect these cancer cells. The combined targeted therapy and detection strategy described here could be exploited for liver cancer therapy and diagnostics, with a moderate safety profile, and may also be a potential tool for other types of cancer.