Iron deficiency is the most common and widespread nutritional disorder worldwide. The automated haematology analyser XN-30 (Sysmex, Kobe, Japan) was developed to detect malaria-infected red blood cells (RBCs) in human blood samples using flow cytometry. The optical system of the analyser detects autofluorescence (AF)-emitting RBCs containing iron-deficient haem groups and would aid in the diagnosis of anaemia resulting from iron deficiency. Here, an RBC-optical (RBO) channel was devised and implemented on the analyser. In vitro analyses showed that the analyser detected AF-emitting RBCs treated with 5-aminolevulinic acid. Furthermore, the analyser detected AF-emitting RBCs in mice fed a low iron diet and infected with a rodent malaria parasite; it could also be effectively used in humans. This study demonstrates that the analyser can quantitatively and reproducibly detect AF-emitting RBCs and measure other haematological parameters, suggesting its usefulness for the initial evaluation of latent iron deficiency anaemia in conjunction with the diagnosis of malaria.