Abstract: Self-assembly films have demonstrated an efficient method to functionalize the surfaces of variously different materials. In this work, we preliminarily explored the modification effect of 10,12-pentacosadiynoic acid (PCDA) on the optical properties of monolayer molybdenum disulfide (MoS₂) grown on a rutile titanium dioxide (r-TiO₂) (110) single crystal surface. Atomic force microscopy (AFM) characterizations directly revealed that the PCDA molecules self-assemble into the same lamella structure as on pure MoS₂, which can be further polymerized into conductive polydiacetylene (PDA) chains under ultraviolet light (UV) irradiation. Detailed photoluminescence (PL) measurements observed clearly increased luminescence of negative trions (A⁻) yet decreased total intensities for MoS₂ upon adding the PCDA assembly, which is further enhanced after stimulating its polymerization. These results indicate that the PCDA assembly and its polymerization have different electron donability to MoS₂, which hence provides a deepened understanding of the interfacial interactions within a multicomponent system. Our work also demonstrates the self-assembly of films as a versatile strategy to tune the electronic/optical properties of hybridized two-dimensional materials.