<p>Membrane processes are one of the most promising separation and extraction technologies in modern industries due to their multiple advantages, including low energy consumption, operational simplicity, and scalability for commercial applications. Currently, these processes, particularly affinity polymer membranes, are a significant research area for selective separations. This study investigates a novel affinity polymer membrane containing Dibenzo-18-crown-6 for the extraction and separation of alanine and tryptophan from aqueous solutions. The membrane was characterized and evaluated for these amino acids individually and in mixtures. In addition to quantitative evaluation, qualitative analysis indicated that the incorporation of DB18C6 modifies the membrane structure and enhances specific interactions between the carrier and amino acids, which influence the extraction process and selectivity. Thermodynamic and activation parameters, including enthalpy (<Emphasis Type="BoldItalic">ΔH</Emphasis>_{<Emphasis Type="BoldItalic">th</Emphasis>}), activation energy (<Emphasis Type="BoldItalic">E</Emphasis>_{<Emphasis Type="BoldItalic">a</Emphasis>}), transition state enthalpy (<Emphasis Type="BoldItalic">ΔH</Emphasis>^{<Emphasis Type="BoldItalic">≠</Emphasis>}), and transition state entropy (<Emphasis Type="BoldItalic">ΔS</Emphasis>^{<Emphasis Type="BoldItalic">≠</Emphasis>}), were determined to elucidate the substrate extraction mechanisms. Notably, this work demonstrates a higher selectivity for alanine over tryptophan in the mixture separation process, highlighting the novelty of selective separation based on specific extractive agent-substrate interactions. These findings provide valuable insights into the design and optimization of affinity membranes for efficient and selective separation of biomolecules, with strong potential for industrial and commercial applications in pharmaceuticals, food, and chemical processing sectors.</p>