Information about species distribution is crucial to ecological studies. Environmental DNA (eDNA) analysis has recently been used to estimate the distribution of aquatic organisms. Several analytical methods including metabarcoding and species-specific PCR are being used for eDNA analysis. However, when only a few species are targeted, metabarcoding is not cost-effective because of the wasted consumption of read due to amplification of non-target species DNA. On the other hand, speciesspecific PCR requires tests to be repeated multiple times resulting in consuming more DNA templates, and experimental consumables. Here we propose a methodological framework for simultaneously detecting a few species using real-time multiplex PCR. We developed the species-specific primer-probe sets for two species of Japanese medaka (Oryzias latipes and o. sakaizumii), and we used them in the real-time multiplex PCR. In aquarium experiment, even when the species abundances were biased, both species were simultaneously detected in all samples. In a field survey, eDNA analysis and capture survey produced consistent results in all sampling sites, including sites with low fish densities. eDNA analysis using real-time multiplex PCR can be easily applied to other aquatic organisms, enabling a more cost-effective distribution survey of multiple target organisms.