Objective To evaluate the safety of β-glucan biofortified rice through a 28-day oral toxicity test. Methods Weaned SD rats were randomly divided into a control group and low, medium and high dose groups (2.5, 5.0 and 10.0 g/kg BW), 22 in each group, half male and half female. After oral gavage (10 mL/kg BW) and continuous administration for 28 d, eyes were examined before and after the experiment. After the animals were sacrificed, urine routine, hematology, blood biochemistry, organ coefficient measurement and histopathology examination were performed. Results Compared with the control group, there were no significant differences in the appearance, weight, food utilization, organ coefficient, blood biochemistry, and urine routine tests in the sample group. Compared with the control group, the food intake of the male medium, high-dose group and the female high-dose group was statistically different (P<0.05 or P<0.01), but there was no continuity in time, which is considered to be non-toxic significance. The platelets (PLT) of the male medium and high dose groups are statistically different from the control group (P<0.05), but there is no dose-response relationship, only a single index is abnormal, and both are within the laboratory reference value range. Within, it is considered to have no toxicological significance; the pathological examination of the high-dose group showed no pathological changes related to the test substance. Conclusion Under the conditions of this experiment, short-term oral intake of β-glucan bio-fortified rice showed no toxic effects, but the safety of long-term consumption needs further research.