The dissymmetric Mannich reaction of benzothiazol- β-amino esters is of great importance for exploring effective enantioisomer with good bioactivity. The mechanism for Mannich reaction of benzothiazol- β-amino esters catalyzed by simple chiral quinine organocatalyst was investigated through a combination of experiment with theoretical approaches (DFT). With solvent effect taken into consideration, transition states TS (S or R) were confirmed with potent strategy of hybrid density functional M06-2X at the level of 6-311G(d, p) basis set. The key feature of dual activation mechanism lies in the formation of one hydrogen bond O (12)—H (25)—N (57) related to quinine hydroxyl (Cat) and benzothiazol imines (EI) N (57) and the other hydrogen bond N(1)—H(90)—O(79) formation related to tertiary amine of quinine (Cat), by which diethyl malonate is able to be activated into enolic Nu simultaneously. The result obtained through energetic calculation was identified further by IR vibrating frequency to convince of transition state attained to be accurate. As a comparison of (R)TS pathway with (S)TS pathway in potential energy profile, it enables to elaborate that (S) TS pathway executes to afford unique enantioisomer (S). At the same time, one of reacting factors was optimized for increasing enantio-and distereoselectivity using DFT calculations, that is reacting temperature. The lower the temperature is going on, the more the enantio-and distereoselectivity are upgraded.