Two-dimensional organic-inorganic perovskites have garnered extensive interest owing to their unique structure and optoelectronic performance. However, their loose structures complicate the elucidation of mechanisms and tend to cause uncertainty and variations in experimental and calculated results. This can generally be rooted in dynamically swinging spacer molecules through two mechanisms: one is the intrinsic geometric steric effect, and the other is related to the electronic effect via orbital overlapping and electronic screening. Herein, we design three types of spacer molecules, phenyl methyl ammonium (PMA), thiophene methyl ammonium (THMA), and furan methyl ammonium (FUMA), that adopt different aromatic units. We examine the influence of different aromatic spacers on the structural properties of the inorganic layer of the perovskite based on first-principles calculations and find that a marginal change in the aromatic ending group in the spacer ligand would trigger significant changes in the octahedral inorganic layer. We predict that using THMA and FUMA can improve the stability and increase the size of crystal domains because of enhanced binding between the organic and inorganic layers. Compared to the prototype phenyl-based perovskite (PMA)2PbI4, the thiophene-based perovskite (THMA)2PbI4 exhibits states closer to the band edge, thus boosting carrier transport across inorganic and organic layers. Compared with the perovskite using PMA as a spacer cation, the THMA-based perovskite demonstrates a higher dielectric constant and smaller exciton binding energy, suggesting that THMA is more suitable as an organic spacer and a good passivation agent in 3D perovskites. The difference in the screening ability of the molecules induces varying interlayer excitonic binding energy. Our work provides theoretical grounds for the engineering of spacer molecules toward high-efficiency light conversion of mixed perovskites.