The invention relates to a preparation method of a TC16 titanium alloy wire with superfine grain structures. The method comprises the following steps: carrying out surface treatment on a TC16 titaniumalloy wire blank, the alpha phase and beta phase dimensions of which are about 1-2[mu]m, after hot working deformation; and carrying out multi-pass room temperature roll-die drawing deformation withthe accumulated deformation reaching 75-90% to obtain the superfine grain TC16 titanium alloy wire, the alpha phase and beta phase average dimensions of which are smaller than 0.5[mu]m. According to the method, phenomena such as restoration and recrystallized softening are seldom generated when the TC16 titanium alloy grains are refined as the alloy is deformed at room temperature, so that the grain refining effect is remarkable. The preparation method provided by the invention is particularly suitable for industrially preparing the superfine wires, is simple in production step and has a goodapplication prospect.