Juvenile yellowtail Seriola quinqueradiata follow floating algae in the East China Sea and have been caught for natural nursery stocks of mariculture in Japan. However, we have still limited knowledge of prey sources supporting their early growth. Here, we clarify prey organisms and sources of the juvenile fishes that appeared in the Satsunan area, southern Japan based on stable isotope ratios and metabarcoding analysis. Stable isotope ratios of carbon and nitrogen for yellowtail juveniles were equivalent to those for zooplankton and fish larvae that appeared in the Kuroshio and its neighboring waters. The most predominant and frequently appearing prey revealed by metabarcoding analysis was calanoid copepods. The next in importance was poecilostomatoid copepods for smaller juveniles and appendicularians and chaetognaths for larger juveniles. A positive correlation was found for the juvenile growth index to the contribution of calanoid copepods of their gut-content DNA. These results suggest that juvenile growth of yellowtail that appeared in the Satsunan area is supported by not benthic animals on floating seaweeds but by zooplankton in the water column.