Modifying transmembrane channels is essential for enhancing functionality. Current modification methods often require chemical reactions or protein engineering, which can increase technical complexity and workload. The inner transmembrane region of MscS can bind lipid molecules, referred to as pore lipids, offering an opportunity for fine-tuning channel properties and improving sensing performance. We leveraged lipid-like molecules, specifically detergents, to replace pore lipids, introducing specific chemical groups into the MscS channel. Using Cryo-EM, we determined the structure of MscS from Pseudomonas aeruginosa, demonstrating that DDM bind stably at the entrance of the hydrophobic pathway. Single-channel recordings confirmed that detergent substitution within the pore alters channel conductance and gating behavior. Molecular dynamics simulations further revealed that detergent modifications adjust the tilt of the TM1-TM2 helices, influencing the channel state. By modifying MscS with different detergents, we tailored its properties, enabling selective detection of target molecules at the single-molecule level. This approach capitalizes on the intrinsic characteristics of MscS and lipid-like molecules, providing a convenient and efficient method for nanopore modification without complex chemical reactions or protein recombination.