For targeted gastric carcinoma therapy, hyaluronic acid (HA)-modified layer-by-layer nanoparticles (NPs) are applied for improving anticancer treatment efficacy and reducing toxicity and side effects. The aim of this study was to develop HA-modified NPs for the co-loading of irinotecan (IRN) and 5-fluorouracil (5-FU). A novel polymer–chitosan (CH)–HA hybrid formulation (HA–CH–IRN/5-FU NPs) consisting of poly( d, l-lactide- co-glycolide) (PLGA) and IRN as the core, CH and 5-FU as a shell on the core and HA as the outmost layer was prepared. Its morphology, average size, zeta potential and drug encapsulation ability were evaluated. Human gastric carcinoma cells (MGC803 cells) and cancer-bearing mice were used for the testing of in vitro cytotoxicity and in vivo antitumor efficiency of NPs. HA–CH–IRN/5-FU NPs displayed enhanced antitumor activity in vitro and in vivo than non-modified NPs, single drug-loaded NPs and drugs solutions. The results demonstrate that HA–CH–IRN/5-FU NPs can achieve impressive antitumor activity and the novel targeted drug delivery system offers a promising strategy for the treatment of gastric cancer.