Advances in cell immunotherapy underscore the need for effective methods to produce large populations of effector T cells, driving growing interest in T-cell bioprocessing and immunoengineering. Research suggests that T cells demonstrate enhanced expansion and differentiation on soft matrices in contrast to rigid ones. Nevertheless, the influence of antibody conjugation chemistry on these processes remains largely unexplored. In this study, we examined the effect of antibody conjugation chemistry on T cell activation, expansion and differentiation using a soft and biocompatible polydimethylsiloxane (PDMS) platform. We rigorously evaluated three distinct immobilization methods, beginning with the use of amino-silane (PDMS-NH₂-Ab), followed by glutaraldehyde (PDMS-CHO-Ab) or succinic acid anhydride (PDMS-COOH-Ab) activation, in addition to the conventional physical adsorption (PDMS-Ab). By employing both stable amide bonds and reducible Schiff bases, antibody conjugation significantly enhanced antibody loading and density compared to physical adsorption. Furthermore, we discovered that the PDMS-COOH-Ab surface significantly promoted IL-2 secretion, CD69 expression, and T cell expansion compared to the other groups. Moreover, we observed that both PDMS-COOH-Ab and PDMS-NH₂-Ab surfaces exhibited a tendency to induce the differentiation of naïve CD4⁺ T cells into Th1 cells, whereas the PDMS-Ab surface elicited a Th2-biased immunological response. These findings highlight the importance of antibody conjugation chemistry in the design and development of T cell culture biomaterials. They also indicate that PDMS holds promise as a material for constructing culture platforms to modulate T cell activation, proliferation, and differentiation.