Small molecules are generally not thought to be assayed by direct sandwich immunoassay, because they cannot be simultaneously bound by two antibodies. In the present study, we demonstrated that small molecules consisting of two epitopes separated by as little as 2.4 Å could be detected by sandwich immunoassay. Through experimental and theoretical study, the success of a sandwich immunoassay was found to be highly probable for two epitopes with an analyte distance above 8.8 Å. These results confirmed the feasible formation of ternary complexes by the association of two mAbs with a very short epitope distance. Notably, the high flexibility of extra-long epitope distances may decrease the stability of these antibody-analyte-antibody complexes, resulting in the low sensitivity of the sandwich immunoassay. Besides, antibodies with deep or shallow binding cavities induced by haptens with extra-long or short spacer arms are detrimental to the validation of sandwich immunoassays for small molecules. We believe that the data presented here can be of general interest in the field of immunoassays, providing a reliable reference for the successful establishment of sandwich immunoassays for small molecules with improved sensitivity, detection range, and specificity.