Rapid and accurate diagnosis of Clostridium difficile infections (CDI) is crucial for patient treatment, infection control and epidemiological monitoring. As an important antigen, glutamate dehydrogenase (GDH) has been proposed as a preliminary screening test target for CDI. However, current assays based on GDH activity or GDH immunoassays have suboptimal sensitivity and specificity. Herein, we describe the selection and characterization of single-stranded DNA aptamers that specifically target GDH. After 10 rounds of selection, high-throughput sequencing was used to identify enriched aptamer candidates. Of 10 candidates, three aptamers for GDH were identified. Gel shift assays showed that these aptamers exhibited low nanomolar affinities. One aptamer was optimized based on structural analysis and further engineered into a structure-switching fluorescence signaling aptamer, wherein desorption from reduced graphene oxide (RGO) upon binding of GDH led to an increase in fluorescence emission. This method allowed for quantitative detection of GDH with a detection limit of 1 nM, providing great potential for its further application in CDI diagnosis.