Nanotherapeutics are destination-aware drug cargo which help improving the treatment duration and reducing drug side effects. Biodegrability and biocompatibility of chitosan nanoparticles (ChNPs) are progressively used for biomedical applications. In this work, ChNPs were prepared by ionotropic gelation technique through electrostatic interaction between chitosan chains (positively charged) and non-toxic polyanions tripolyphosphate (TPP) (negatively charged). The ratio between chitosan and TPP was found to have a crucial role on the size, the poly dispersity index (PDI), and the zeta potential of the prepared ChNPs. Different volumetric ratios of Ch:TPP (1:1, 2:1, and 3:1) were prepared. The optimally prepared ChNPs were used for encapsulating the anticancer drug, 5- Fluorouracil (5-FU) forming (5-FU-ChNPs). ChNPs and 5-FU-ChNPs gave hydrodynamic diameter of 140.4 nm and 293 nm respectively, which were confirmed by transmission electron microscope (TEM). Cytotoxic assay was carried out on hepatocellular carcinoma (HepG2) cell line and it revealed the effectiveness of laser-irradiated 5-FU-ChNPs compared with in absence of laser irradiation. The 5- FU-ChNPs exhibited the desired light absorption causing hydrolysis and secession of the polymer chains at the tumor site. This led to efficient destruction of the cancer cells and verified the effectiveness of 5-FU-ChNPs as drug delivery system assisted by laser irradiation.