Objective To establish a model of oral mucositis in golden hamsters induced by 5-fluorouracil combined with mechanical scratching. Methods Male golden hamsters were divided into a control group and model group.Animals in the model group were intraperitoneally injected with 60 mg/ kg and 40 mg/ kg of 5-fluorouracil (5-FU) on the first and second days, and the right cheek pouch was slightly scratched by an 18-gauge needle on the fourth day. The control animals were intraperitoneally injected with an equal volume of normal saline on the first and second days. After the treatment, the pathological changes of the right cheek pouch of each animal were scored every day and the body weight and the mean daily food intake of each animal were recorded. The level of proinflammatory cytokines TNF-α and IL-1β in the serum was measured by ELISA. The right cheek pouch tissue was removed for pathological examination on days 8 and 14 after the treatment, and the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in the right cheek pouch tissue were determined by biochemical analysis. Results The model group developed an ulcer on the surface of the right cheek pouch on the third day after treatment, and the macroscopic scores of right cheek pouch were the highest compared with the control group. The mean daily food intake was significantly reduced and the body weight was decreased within 8 days after the treatment in the model group. On days 8 and 14 after modeling, the histological structures of the right cheek pouch tissues were damaged and had an inflammatory reaction. The levels of TNF-α and IL-1β in the serum of golden hamsters were significantly increased, the MDA content in the cheek pouch tissue was significantly increased, and the SOD activity was reduced on days 8 and 14. Conclusions 5-fluorouracil combined with mechanical scratching induces the formation of oral mucositis in golden hamsters. The inflammatory response is marked within 8 days after the treatment, and the most obvious is on the third day. Therefore, this model can be used to study the pathogenesis of oral mucositis and for the development of therapeutic drugs.