The invention discloses an alpha isomer impurity of regadenoson and a preparation method and a medicinal use thereof. The impurity is obtained through oriented synthesis; 2,6-dichloropurine and beta-D-1,2,3,5-tetra-o-acetyl-d-ribose are taken as starting raw materials; a salt of alpha 2-chloroadenosine is obtained by concentration, ammonolysis, salifying and purification; the regadenoson alpha isomer impurity is obtained directly through hydrazinolysis, concentration and ammonolysis without dissociation. An isomer is taken as a regadenoson impurity reference substance, so that the content of the impurity produced in synthesis can be effectively identifies, thereby raising the medicine quality standard of regadenoson.