Abstract: To characterize the diet and biology of juvenile Mystus guttatus, and to provide a scientific basis for its resource conservation and population restoration, we applied DNA macro barcode technology to analyze the species characteristics of stomach content of M. guttatus juvenile and water body in the river channel of M. guttatus conservation farm in Luodian County, Guizhou Province. The results show that: 1) A total of 109 species of organisms were identified in the stomach contents of M. guttatus juvenile, belonging to 15 phyla and 12 major categories. On phylum level, the relative abundance of Arthropods was the highest, followed by Rotifera, Chlorophyta and Chordata. On genus level, the relative abundance of Sinodiaptomus was the highest, followed by Brachionus, Barbus and Paralamyctes. 2) A total of 193 species belonging to 18 phylum were obtained in the river channel where M. guttatus juvenile fish lived. On phylum level, the relative abundance of Arthropods was the highest, followed by Chlorophyta. On genus level, the relative abundance of Sinodiaptomus was the highest, followed by Chlamydomonas. Other genus with higher relative abundance were Brachionus, Tintinnidium and Monoraphidium. Arthropoda, Rotifera and Chlorophyta were relatively abundant groups in the stomach content of M. guttatus juvenile and water body. The relative abundance of Chordate was low, ranking 15^th in the water body, and 4^th in the stomach content. The results indicate that the dietary selection of M. guttatus juvenile is influenced by both the availability of food in water environment and the preference of juvenile. From the perspective of energy balance benefits, the feeding process of M. guttatus juvenile follows the principle of obtaining maximum benefits with minimal energy input, which is beneficial for its survival and growth.