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Abstract Background The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study. Methods Test zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay. Results Screening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a K D of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD. Conclusion The reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests. Graphical Abstract The assembly of µPAD sensors using APTEC assay principles for the detection the malarial biomarker, lactate dehydrogenase enzymes from Plasmodium falciparum (PfLDH). The aptamers immobilized at the test zones capture the PfLDH in samples. After washing the unbound sample components from the zones, Malstat assay reagents are added for colour development, proportional to the amount of captured PfLDH.