To reduce the toxicity of methotrexate (MTX) and increase the targeting of nanoparticles, the MTX-loaded chitosan (CS) covalently bonded with folic acid (FA) nanoparticles were prepared, and sodium tripolyphosphate was used as the cross-linking agent. FA was successfully conjugated to CS confirmed by H-1-NMR and Fourier transform infrared spectrometer (FT-IR). The prepared FA-CS nanoparticles were characterized by FT-IR spectroscopy to confirm the cross-linking reaction between FA-CS and cross-linking agent. X-ray diffraction was performed to reveal the crystalline nature of the drug after encapsulation. The average diameters of the nanoparticles ranged from 293.9 +/- A 24.2 to 401.5 +/- A 20.4 nm with a narrow particle size distribution. In vitro release pattern in phosphate buffer saline (pH 6.8) indicated that the characteristics of the MTX-loaded nanoparticles appeared to have an initial burst effect and followed by a slow, sustained drug release. FA or low molecular weight FA conjugate fragments were also released from the nanoparticles, which might have potential to reduce toxic effects of MTX within the body.