BackgroundHigh-quality malaria diagnosis is essential for effective treatment and clinical disease management. Microscopy and rapid diagnostic tests are the conventional methods performed as first-line malaria diagnostics in non-endemic countries. However, these methods lack the characteristic to detect very low parasitaemia, and accurate identification of thePlasmodiumspecies can be difficult. This study evaluated the performance of the MC004 melting curve-based qPCR for the diagnosis of malaria in routine clinical practice in non-endemic setting.Methods and resultsWhole blood samples were collected from 304 patients with clinical suspicion of malaria and analysed by both the MC004 assay and conventional diagnostics. Two discrepancies were found between the MC004 assay and microscopy. Repeated microscopic analysis confirmed the qPCR results. Comparison of the parasitaemia of nineteenPlasmodium falciparumsamples determined by both microscopy and qPCR showed the potential of the MC004 assay to estimate the parasite load ofP. falciparum. EightPlasmodiuminfected patients were followed after anti-malarial treatment by the MC004 assay and microscopy. The MC004 assay still detectedPlasmodiumDNA although no parasites were seen with microscopy in post-treatment samples. The rapid decline inPlasmodiumDNA showed the potential for therapy-monitoring.ConclusionImplementation of the MC004 assay in non-endemic clinical setting improved the diagnosis of malaria. The MC004 assay demonstrated superiorPlasmodiumspecies identification, the ability to indicate thePlasmodiumparasite load, and can potentially detect submicroscopicPlasmodiuminfections.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-023-04617-z.