Two sets of isomeric organic dyes with n-hexyl (DH and AH) or 2-ethylhexyl (DEH and AEH) groups substituted at the spacer part have been designed and straightforwardly synthesized via a facile and selective synthetic route. The structure difference between the isomers stands at the position of the incorporated alkyl chains which are introduced into the terthiophene spacer close to the donor (D) or anchor (A) side. The relationship between the isomeric structures and the optoelectronic properties are systematically investigated. It is found that, in the D series dyes, the alkyl group is much closer to the aromatic donor moiety, which brings about strong steric hindrance and therefore causes a remarkable twist in the molecular skeleton. In contrast, a more planar chemical structure and more effective π-conjugation are realized in the A series dye isomers. Consequently, the A series isomeric dyes demonstrate bathochromically shifted absorption bands, resulting in the improved light-harvesting capability and enhanced photo-generated current. However, the D series isomeric dyes with more twisted molecular skeleton have suppressed the intermolecular interactions and retarded the charge recombination more efficiently, which induces higher open-circuit photovoltage. Combining the two effects on the performance of the fabricated dye-sensitized solar cells (DSSC), the influence from the short-circuit photocurrent plays a more significant role on the power conversion efficiency (η). As a result, isomer AEH-based DSSC with quasi-solid-state electrolyte displays the highest η of 7.10% which remained at 98% of the initial value after continuous light soaking for 1000 h. Promisingly, a η of 8.66% has been achieved for AEH-based DSSC with liquid electrolyte containing Co(II)/(III) redox couple. This work presents the crucial issue of molecular engineering and paves a way to design organic sensitizers for highly efficient and stable DSSCs.