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Enzyme-linked immunosorbent assays (ELISAs) allow for the testing of large numbers of samples within a short time frame. We tested the sensitivity and specificity of a histidine-rich protein 2 (HRP2)-based, commercially available ELISA antigen detection assay for Plasmodium falciparum (Malaria Antigen CELISA; Cellabs, Sydney, Australia). A total of 700 whole blood samples obtained from symptomatic outpatients of malaria clinics along the Thai–Myanmar border were tested relative to blinded duplicate expert microscopy adjusted with species-specific polymerase chain reaction (PCR). PCR-adjusted microscopy showed that 79 (11.3%) were infected with P. falciparum, 118 (16.9%) with P. vivax, 1 (0.1%) with P. malariae, 7 (1.0%) with mixed infections (P. falciparum and P. vivax), and 495 (70.7%) were negative. The geometric mean parasite density for P. falciparum was 7547/μL (range: 12–363,810/μL). The overall sensitivity of the HRP2 ELISA for P. falciparum malaria was 98.8% (95% CI, 93.6–100%) and the specificity was 100% (95% CI, 99.5–100%). The positive and negative predictive values for the ELISA were 100% (95% CI, 96.5–100%) and 99.8% (95% CI, 99.1–100%), respectively. The results for P. falciparum were clearly superior to expert microscopy alone, particularly in mixed infections. Microscopy combined with ELISA reaches a sensitivity and specificity similar to PCR-adjusted microscopy for the diagnosis of P. falciparum while being considerably less expensive and faster. We conclude that ELISA serves as an excellent tool to augment microscopy as the gold standard for P. falciparum diagnosis in research settings and should be further evaluated for screening in blood banks.