AbstractAptamers are ligand-binding RNA or DNA molecules and have been widely examined as biosensors, diagnostic tools, and therapeutic agents. The application of aptamers as biosensors commonly requires an expression platform to produce a signal to report the aptamer-ligand binding event. Traditionally, aptamer selection and expression platform integration are two independent steps and the aptamer selection requires the immobilization of either the aptamer or the ligand. These drawbacks can be easily overcome through the selection of allosteric DNAzymes (aptazymes). Herein, we used the technique of Expression-SELEX developed in our laboratory to select for aptazymes that can be specifically activated by low concentrations ofl-phenylalanine. We chose a previous DNA-cleaving DNAzyme known as II-R1 as the expression platform for its low cleavage rate and used stringent selection conditions to drive the selection of high-performance aptazyme candidates. Three aptazymes were chosen for detailed characterization and these DNAzymes were found to exhibit a dissociation constant forl-phenylalanine as low as 4.8 μM, a catalytic rate constant improvement as high as 20 000-fold in the presence ofl-phenylalanine, and the ability to discriminate against closely relatedl-phenylalanine analogs includingd-phenylalanine. This work has established the Expression-SELEX as an effective SELEX method to enrich high-quality ligand-responsive aptazymes.