Herein, we report a sensitive and selective enzyme-linked aptamer-based sandwich assay (ELASA) to detect Plasmodium falciparum lactate dehydrogenase (PfLDH), which is an attractive biomarker for malaria diagnosis and antimalarial medication. We performed the sandwich assay with a single aptamer sequence, called 2008s, owing to the structural properties of the PfLDH tetramer instead of using a conventional sandwich assay with two different aptamers (or antibodies) for capturing and probing a target molecule. First, the biotinylated PfLDH aptamer was linked with immobilized streptavidin on a microwell plate for binding flexibility, and then PfLDH was bound to the aptamer. Next, a horseradish peroxidase-conjugated aptamer of the same sequence was used to analyze PfLDH quantitatively. Using this approach, the limit of detection (LOD) of PfLDH with the naked eye was 100 ng mL⁻¹, and the LOD and limit of quantification from the absorbance measurements were 34.9 ng mL⁻¹ and 95.5 ng mL⁻¹, respectively, based on PfLDH spiked blood samples. Our proposed method selectively binds PfLDH, not human lactate dehydrogenase. Therefore, this method may be a valuable tool for diagnosing, monitoring, and quarantining malaria cases easily and rapidly.