The size effect of Pd catalyst and CO as a selective accelerator in C2H2 semi-hydrogenation over Pd catalyst are two main factors to affect catalytic performance. In this work, density functional theory calculations were used to unravel the roles of Pd cluster size and introduced CO over the anatase TiO2 supported Pdn (n = 2, 3, 4, 7, 13) clusters in tuning catalytic performance of C2H2 semi-hydrogenation. The results show that as the increasing of Pdn cluster size over Pdn/TiO2 catalysts, C2H4 selectivity generally presents a volcanic-type curve, and the activity of C2H4 and green oil presents an inverted volcanic-type curve. Compared with Pdn/TiO2 catalysts, the introduction of CO greatly improves C2H4 selectivity over Pd2/TiO2 and Pd3/TiO2 catalysts, enhances C2H4 formation activity over Pdn/TiO2 (n = 3, 4, 7, 13), and decreases green oil production over Pd2/TiO2 and Pd13/TiO2 catalysts. Meanwhile, the activation free energy of C2H4 hydrogenation and C2H3 coupling reaction can be used as the evaluate index to quantitatively predict the selectivity of C2H4 and green oil, respectively. The different geometric and electronic effects induced by the introduction of CO could tune catalytic performance. The screened Pd4/TiO2 and Pd2CO/TiO2 catalysts are the most suitable catalysts in the Pdn/TiO2 and PdnCO/TiO2 catalysts, respectively. The relationship of Pdn cluster size and CO introduction with C2H4 selectivity and formation activity would provide valuable structural clue for the construction of C2H2 semi-hydrogenation catalyst with highly catalytic performance.