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We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0 parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35 min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5–8 h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous., Graphical abstract Magnetic microparticle-based immunoassay reproducibly detects two malarial biomarkers in under an hour with detection limits an order of magnitude better than commercially available kits.fx1, Highlights • Rapid, magnetic microparticle-based detection of pLDH and PfHRPII from one sample. • Detection of both biomarkers is critical in the context of malaria elimination. • Total incubation time of 35 min. • LODs an order of magnitude below commercial ELISA kits, within asymptomatic regime. • Reproducible across users over time, and simple enough for novice users.