The acquisition of Apocynum venetum mutant library is an effective way to innovate germplasm resources artificially, and it is also the basis of varieties breeding and functional genome research. In this study, wild A. venetum seeds collected in Dongying were used to construct mutant library. Pre-experiment results indicated that 0.7% ethylmethylsulfone (EMS) solution was the LD50 for seeds of A. venetum treated for 13 h. A mutant library of 1452 individual plants was constructed with the treatment in this concentration and duration. The agronomic traits and total flavonoid contents of the M1 population were screened. A total of 125 plants with abnormal agronomic traits were obtained, including 8 albinos, 23 leaf form variants, 16 yellow-striped leaf variants, 32 branching seedling variants, and 46 leaf position variants. The mutation frequencies were 0.55%, 1.6%, 1.1%, 2.2% and 3.2%, respectively.Moreover, the total flavonoids in two plants were significantly higher and in one plant was lower than that in the wild type. The flavanone 3-hydroxylase gene (F3H) involved in the flavone metabolism pathway was sequenced in the mutant library. The results showed that one in every 6.7 plants appeared as an F3H gene mutant. The mutant library of A. venetum constructed in this study showed abundant phenotypic variations, which provided good materials for selection of new germplasm, varieties breeding and gene function research in A. venetum.