The invention discloses an establishment method of a horsetail beefwood callus regeneration system. The method comprises the steps that tender branches of horsetail beefwood seedlings are used as explants, and the explants are inoculated to a culture medium to obtain swollen stems, wherein the culture medium is prepared from 1/2 MS, 3% of sucrose, 0.1 mg/L of TDZ and 0.1 mg/L of NAA, and pH is 5.7+/- 0.1; the swollen stems are taken and moved to a callus induction culture medium, and callus is obtained, wherein the callus induction culture medium is prepared from 1/2 MS, 3% of sucrose, 0.5 mg/L of 6-BA and 0.1 mg/L of TDZ, and pH is 5.7 +/- 0.1; adventitious buds and taken roots are obtained by inducing the callus, and horsetail beefwood seedlings are obtained. According to the method, the horsetail beefwood callus induction culture is divided into two steps, the swollen stems are firstly obtained through culture with a specific swelling induction culture medium, and then the callus is obtained through culture with the specific callus induction culture medium so that the regeneration system high in conversion rate can be obtained, and the period for obtaining the regeneration system can be obviously shortened.