In the present study, a novel silane coupling agent, designated INSi, was synthesized via a facile synthetic route, incorporating indole-functional moieties. This agent was further employed for the surface modification of homemade silica nanomicrospheres (SMPs). The ensuing nanomicrosphere composite, denoted as SiO2@IN, exemplified pronounced interfacial π-π interactions. Optimization of the reaction conditions was conducted using the response surface optimization technique. Subsequent validation of interfacial π-π interactions was accomplished through a synergistic approach, integrating theoretical calculations and comprehensive analyses of spectral and morphological attributes exhibited by the SiO2@IN.