Pure organic two-photon excited room temperature phosphorescent (RTP) materials have attracted great attention for time-resolved imaging due to their long emission lifetime and high resolution. The materials with an aromatic carbonyl group exhibit aggregation-induced emission (AIE) and RTP characteristics simultaneously. Here, we deeply explored the nature of aggregation-induced phosphorescence (AIP), especially the relationship between molecular configuration and optical properties. It was found that aggregation effect can suppress geometrical vibrations and regulate energy difference between S1 and T1. The aromatic carbonyl group plays significant roles in changing electronic configuration, resulting in large Stokes shift and spin-orbit coupling. It also leads to small transition dipole moment, decreasing two-photon absorption cross section and radiative decay rate. To improve two-photon absorption properties, we further designed a π-conjugated compound with large two-photon absorption cross section in the biological window (36.40 GM/656 nm) and AIP characteristics, which is a potential material in the application of time-resolved two-photon excited imaging.