Synthetic α-helical nanopores offer a powerful platform for tuning pore geometry and functionality through precise molecular chemical modifications for various applications in nanobiotechnology and synthetic chemical biology. Here, we designed a synthetic α-helical peptide pore pPorA, derived from the porin PorACj that autonomously assembles into octameric monodisperse pores. By incorporating either natural or unnatural amino acids at a specific position, we modulate the pore size and conductance, generating two distinct conductance states despite the identical octameric subunit composition. This established the structural flexibility of α-helical pores. Larger pores with higher conductance can sense bulky cyclic sugars and PEGylated molecules, while smaller pores with lower conductance selectively detect linear peptides or small molecules. The larger pores were explored for sensing α-Synuclein, a presynaptic cytosolic protein involved in neurotransmitter release, whose aberrant aggregation is linked to Parkinson’s disease. Using these pores, we were able to effectively detect the distinct unfolding and translocation of α-Syn and its pathological mutants at the single-molecule level. These engineered pores were also utilized to detect distinct conformations of another intrinsically disordered protein, Humanin, which exhibits neuroprotective and anti-apoptotic effects but loses these protective functions upon mutations. These findings demonstrate the versatility of site-specific modifications in α-helical peptide pores, enabling the design of tunable sensors with broad molecular sensing capabilities. This work lays the groundwork for developing peptide-based sensors with applications in biosensing, complex single-molecule detection and disease diagnosis.