The present study aimed to formulate 5-fluorouracil loaded cross linked chitosan nanoparticles based on chemical cross-linking of low molecular weight chitosan with glutaraldehyde by reverse micelles technique as 5-FU is less hydrophobic, relatively potent, has a shorter half-life, is rapidly metabolized, less tolerated, and has low oral bioavailability; therefore, we aimed to formulate potential nanocarriers of 5-FU for efficient drug delivery to specific targeted areas of action, reduce oral toxicity, improve tolerability and therapeutic outcomes of 5-FU, in a restricted fashion to enhance the bioavailability of 5-FU. Nanoparticles were formulated by the reverse micelle method based on the chemical cross-linking of glutaraldehyde (25% aqueous solution) into a w/o emulsion in different ratios. LMWCH-NPs were characterized for post-formulation parameters by mean particle size, zeta potential, %age yield, loading/entrapment efficiency, Fourier transform infrared spectroscopy (FTIR), DSC/TGA, TEM, PXRD, drug release at pH 1.2, and pH 7.4. 5-FU loaded NPs showed a size range (198 nm-200 nm) and zeta potential (−39mV to −41mV), which ensured mechanical stability and increased retention time in blood vessels by the sustained release properties of biodegradable nanocarrier drug delivery systems. % age yield showed the range 92% to 96% while % LC ranged 2.0% to 3.4% and %EE ranged 40% to 43%. The TEM images showed spherical nanoparticles. FTIR revealed the compatibility between the drug and the cross-linked polymer. DSC/TGA ensured the thermal stability of the drug, while the solid-state stability of the drug-loaded cross-linked chitosan nanoparticles was evaluated by powder X-ray diffraction (PXRD) analysis. Drug release studies were performed using the dialysis bag technique at both pH (1.2 and 7.4) to mimic the gastrointestinal tract. Highly stable NPs displayed targeted release in phosphate buffer pH 7.4 at 37°C. Fickian diffusion was the predominant release with an R2 value of 0.9975-0.9973—and an N value 0.45-0.53. Prepared nanoparticles are inert, biodegradable, and biocompatible drug delivery systems for sustained release of 5-FU with maximum therapeutic efficacy and bioavailability.