The invention provides TC21 titanium alloy powder for laser 3D printing and preparation and using methods. The powder is prepared from, by mass, 5.5%-6.7% of Al, 1.7%-2.4% of Sn, 1.7%-2.4% of Zr, 2.4%-3.1% of Mo, 1.0%-1.8% of Cr, 1.8%-2.2% of Nb, 0.004%-0.18% of Fe, 0.004%-0.01% of C, 0.002%-0.009% of N, 0.003%-0.01% of H, 0.08%-0.1% of O and the balance Ti. The preparation method comprises the steps that TC21 titanium alloy is machined into an electrode and then rotates and goes down in a smelting chamber, gas acts on the conical tip of a titanium bar when the tip of the titanium bar is bright white, titanium alloy liquid drops are brought into an atomizing chamber through nozzles to be cooled into alloy powder, and the alloy powder is collected. The using method comprises the steps that after the alloy powder with the particle size ranging from 54 micrometers to 180 micrometers and a substrate are pretreated, the as-deposited TC21 titanium alloy with the good strength and toughness is printed by adopting a laser 3D printer, wherein the tensile strength is 1,100-1,150 Mpa, the yield strength is 1,010-1,030 Mpa, and the elongation rate is 7%-8%.