The microscopic examination of Giemsa-stained thin and/or thick blood films (Giemsa microscopy) is the standard method of malaria diagnosis. However, the results of the diagnosis significantly depend on the skills of clinical technicians. Furthermore, sample preparation and analysis are laborious and time-consuming. Therefore, in this study, we investigated if a commercially available fluorescent cell counter, LUNA-FL, was useful for the detection of Plasmodium parasite and the estimation of parasitemia. Whole blood samples from uninfected persons, spiked with P. falciparum-infected erythrocytes, were analysed. Most of the leucocytes and platelets were removed from whole blood samples with SiO2-nanofiber filters set on spin columns. The filtered samples were stained with acridine orange, and automatic detection, as well as counting of erythrocytes and parasites, were performed using LUNA-FL. Whole blood, with various levels of parasites, was analysed by Giemsa microscopy or with LUNA-FL to estimate parasitemia, and a comparative analysis was performed. The coefficient determination value of the regression line was high (R2 = 0.98), indicating that accurate quantitative parasite detection could be performed using LUNA-FL. LUNA-FL has a low running cost; it is compact, fast, and easy to operate, and may therefore be useful for point-of-care testing in the endemic areas.