The application provides a medicine containing methotrexate, its preparation method, pharmaceutical composition and application. The drug includes nucleic acid nanoparticles and methotrexate, and the methotrexate is mounted on the nucleic acid nanoparticles; the nucleic acid nanoparticles include a nucleic acid domain, the nucleic acid domain includes a sequence, b sequence and c sequence, and the a sequence includes a1 sequence or a1 sequence with at least one base insertion, deletion or substitution, b sequence contains b1 sequence or b1 sequence with at least one base insertion, deletion or substitution, c sequence contains c1 sequence or c1 sequence with at least one base sequences of insertions, deletions, or substitutions. The drug containing methotrexate provided by the present application, after the nucleic acid structure domain is modified by the target head, can have better targeting, can deliver methotrexate stably, and has high reliability.