Aims: To prepare 5-fluorouracil (5-Fu) nanoparticles with higher encapsulation efficiency and drug loading, and then investigate interaction with the SGC-7901 gastric cancer cell line. Materials and Methods: Prescription was optimized by orthogonal experiments, the encapsulation efficiency and loading capacity were tested by high-performance liquid chromatography, and inhibition of proliferation by 5-Fu nanoparticles and 5-Fu given to cells for 24, 48 and 72 hours was investigated by methyl thiazolyl tetrazolium assay (MTT). In addition, 5-Fu nanoparticles were labeled by fluorescein isothiocyanate (FITC), and absorption into cells was tested by flow cytometry. Results: The optimal conditions for preparation were concentrations of 5-Fu of 5mg/ml, of $CaCl_2$ of 60 mg/ml and of chitosan of 2 mg/ml. With a stirring speed of 1200rpm, encapsulation efficiency of 5-Fu nanoparticles was $55.4{pm}1.10%$ and loading capacity was $4.22{pm}0.14%$; gastric cancer cells were significantly inhibited by 5-Fu nanoparticles in a time and concentration dependent manner, and compared to 5-Fu with slower drug release, in a certain concentration range, inhibition with 5-Fu nanoparticles was stronger. 5-Fu nanoparticles were absorbed by the cells in line with the concentration. Conclusions: 5-Fu nanoparticles can inhibit growth of gastric cancer cells in vitro to a greater extent than with 5-Fu with good adsorption characteristics, supporting feasibility as a carrier.