Abstract: Biotinylated chitosan nanoparticles (Bio-CS-NP) were prepared for the active delivery to cancer cells and its characterization was investigated in this study. The preparation process included two steps. First, biotinylated chitosan (Bio-CS) was obtained through a reaction between sulfosuccinimidobiotin and chitosan (CS). Second, Bio-CS-NP were prepared by the precipitation of Bio-CS with sodium chloride solution. With a biotin reagent box, the conjugation densities of biotin on the surface of Bio-CS-NP were determined. The morphology and diameter of the nanoparticles were assayed by transmission electron microscope (TEM) and laser light scattering particle analyzer, respectively. The uptake of nanoparticles by human hepotacarcinoma HepG2 cells, for example, Bio-CS-NP and chitosan nanoparticles (CS-NP) without any modification, was quantitatively examined. The results indicated that the conjugation densities of biotin on the surface of Bio-CS-NP were 2.2 biotin CS. Bio-CS-NP were spherical, smooth on the surface. The average diameter was 296.8 nm. The polydispersion index was 0.155. The uptake of Bio-CS-NP by HepG2 cells was much higher than that of CS-NP (P<0.05). It demonstrated that Bio-CS-NP can be applied as a new vehicle to actively deliver anticancer drugs to tumor cells. The method for the determination of biotin was simple and practical.