Molecular wires with asymmetric anchors have garnered considerable interest in the field of molecular electronics. Numerous studies have focused on asymmetrically anchored molecules at both single-molecule and self-assembled monolayer (SAM) levels. However, few studies have investigated how the binding preference of asymmetric anchors towards the substrate affects their quantum transport behavior. In this study, oligo(arylene ethynylene) derivatives with thiol acetate anchors at one terminal and pyridine anchors at the other terminal were used for self-assembly, and gold and single-layered graphene (SLG) were employed as the bottom and top electrodes to form molecular junctions. XPS results indicated that, without deprotecting the acetyl group on thiol acetate, the molecules tended to assemble on the Au surface with either the thiol anchor or pyridine anchor. However, with the deprotection procedure (which transformed the thiol acetate into thiol), almost all molecules tended to assemble on the Au surface with the thiol anchor. Furthermore, quantum transport measurements revealed that both the electron tunnelling efficiency and the energy difference between the electrode Fermi level and the molecular frontier orbital also shifted due to this change in the binding preference. For example, the field effect transistor behaviour of functional SAMs can be switched between ambipolar (where the molecule can be turned on by shifting the gate voltage in either the positive or negative direction, resembling an ambipolar MOS-FET) and unipolar (where the molecule can only be turned on by shifting the gate voltage in the negative direction, resembling an n-type MOS-FET). This study demonstrates that, in addition to molecular structure engineering, molecular electronic functionalities such as tunnelling efficiency and switching behaviour can also be regulated through binding preference control during self-assembly. These findings suggest a new approach for fabricating advanced quantum technology devices.