Objective To elucidate the long-term effect of isoproterenol on inducing anxiety-like behavior under chronic stress in kunming mice. Methods A total of 50 Kunming mice were randomly divided into control, chronic stress, isoproterenol (ISO) group, chronic stress +ICI 118, 551 (ICI) group and ISO+ICI 118, 551(ISO+ICI) group (n = 10 in each group). Control group: normal saline was intraperitoneally injected every day; Chronic stress group: Chronic stress treatment was given every day. ISO treatment group: ISO (5 mg/kg) was injected intraperitoneally every day. Chronic stress +ICI treatment group: ICI (0.2 mg/kg) was injected intraperitoneally every day after chronic stress treatment. ISO+ICI treatment group: ISO(5mg/kg) and ICI(0.2 mg/kg) were given intraperitoneally every day. Results The open arm residence time, the number of entering open arm, the percentage of open arm residence time and the number of entering open arm in the chronic stress group and ISO group were significantly lower than those in the control group (P<0.05). The open-box retention time, percentage of open-box retention time, open-box activity times and shuttle times in the chronic stress group and ISO treatment group were significantly lower than those in the control group (P<0.05). The retention time of dark box was significantly higher than that of the control group (P<0.05). Conclusion After long-term treatment with β-AR agonist isoproterenol, kunming mice developed anxiety-like behavior under chronic stress.