The organic-inorganic hybrid quasi-two-dimensional (quasi-2D) perovskites have attracted increasing attention for solar cell applications due to their improved moisture stability and excellent optoelectronic properties. To achieve a profound understanding and delicate control on the component of perovskite, it is indispensable to know how different spacer cations affect their intrinsic properties. Here, we synthesized a series of quasi-2D perovskite single crystals with different organic spacer cations and conducted a systematic investigation to correlate the optoelectronic behaviors to the spacer cations. We revealed the coupling effect between the π-electron (PEA+) and p orbital of I- from the inorganic framework, which changed the electronic configuration of resultant crystals leading to different band gap and carrier behavior. In particular, the anisotropic carrier mobility was probed quantitatively by space-charge limited current (SCLC) measurement, where PEA2MA2Pb3I10 exhibited the highest in-plane mobility due to the reduced exciton binding energy and the lowest out-of-plane mobility because of the widest organic barrier.