In order to evaluate the safety of recombinant human rhinovirus 3C fusion protease (GST-3C), the toxicity of GST-3C was studied by single-dose toxicity test and immunotoxicity test. Single-dose toxicity test: a drug administration group was set up, and 10 male and 10 female ICR mice were prepared. GST-3C was injected into the tail vein by maximum dose method, and the maximum dose of single dose was 25.25 mg/kg. Another normal saline control group was set up. The symptoms, degree, nature, recovery, and death of ICR mice were observed, and the mice were dissected in time at the end of the observation period to observe the effects of GST-3C on the organs of mice. Immunotoxicity test: 50 mice were randomly divided into five groups (solvent control, cyclophosphamide positive control group, adjuvanted GST-3C low-dose group, adjuvanted GST-3C high-dose group, GST-3C control group without adjuvant). Spleen index, thymus, index and swelling degree were calculated by delayed hypersensitivity method to observe the effect of GST-3C on delayed hypersensitivity. 40 mice were randomly divided into four groups (solvent control, adjuvanted GST-3C low-dose group, adjuvanted GST-3C high-dose group, GST-3C control group without adjuvant). The effect of hemolytic plaque on humoral immune function of mice was evaluated by counting hemolytic plaque test. Single-dose toxicity test showed that compared with the normal saline control group, there was no significant difference in the body weight of the mice in the experimental group, and no abnormal performance was found in the organs of the animals in the administration group. Under the conditions of this experiment, the maximum tolerated dose (MTD) of a single intravenous injection of GST-3C in ICR mice was greater than 25.25 mg/kg. Immunotoxicity test showed that during the administration period, compared with the solvent control group, the body weight of the animals in each group administered with GST-3C did not decrease significantly, and no abnormal changes related to administration were observed beside the animal cage. The results of delayed hypersensitivity test and hemolytic plaque test showed that GST-3C had no inhibitory effect on the cellular immunity and humoral immunity of animals, which provides a basis for evaluating the safety of GST-3C.