Provided is a preparation method of a composition enriched in compound Sp-1. The method performs isomer separation on a reaction product from the first step, and then performs a two-step chemical synthesis, so as to prepare a composition comprising high-purity compound Sp-1, wherein the HPLC purity of the single isomer Sp-1 is 90% or more. Provided is a composition enriched in compound Sp-1. The composition has an inhibitory effect on tumor cell proliferation in vitro significantly higher than that of NUC-1031 and compound Rp-1. Also provided are the use of the composition, and a pharmaceutical composition comprising the composition and at least one pharmaceutically acceptable excipient.