The invention provides a diesel oil anti-wear agent composition and a diesel oil composition and a preparation method thereof. The composition contains (A): 80-97 wt% of fatty acid esters of glycerolwith the carbon number being 10-25, and (B): 3-20 wt% of fatty acid with the carbon number being 10-25. The acid value of the composition is 6-40 mg KOH/g; the content of free glycerol is less than 0.5%; and the content of saturated fatty acid is less than 2.5 wt%. The preparation method of the composition comprises the step of mixing no less than 80 wt% of an ester-type anti-wear agent (fatty acid esters of glycerol) which satisfies the Sinopec purchasing standard and no less than 20 wt% of an acid anti-wear agent (fatty acid) which satisfies the Sinopec purchasing standard. The diesel oil anti-wear agent composition provided by the invention can remarkably improve lubricity of low-sulfur diesel oil.