[Objectives] Amphioxus is a transitional animal from lower invertebrates to higher vertebrates, which plays an extremely important role in the research of origin and evolution of vertebrates. Currently, the family Branchiostomatidae contains three genera: Branchiostoma, Asymmetron and Epigonichthys. In August 2021, when conducted a comprehensive survey in Nanji Islands National Marine Nature Reserve, Zhejiang Province, the author discovered amphioxus which is a national second-class protected animal. [Methods] Compared the specimens (one male ZJ 01♂, one female ZJ 02♀) with other species of amphioxus by comparing their morphological characteristics and measurement data (Fig. 1, Table 2). Phylogenetic analysis was performed on the basis of COI sequences of the samples and other amphioxus to determine the phylogenetic location of the specimens. The Kimura2-Parameter model in MEGA11.0.1 was used to calculate the genetic distances between species of the amphioxus. [Results] We found the specimens were similar with Branchiostoma japonicum in morphological. Based on molecular evidence, the specimens were clustered with the B. japonicum (Fig. 2). Genetic distance estimation based on Kimura2-Parameter model showed that the distance between the specimens and B. japonicum was the closest, ranging from 0.006 to 0.013, the distance much lower than that between other species range from 0.171 to 0.269 (Table 3). [Conclusion] Combined with morphological and molecular biological methods, this species was identified as B. japonicum. According to relevant literatures, the author presumed that amphioxus in Nanji Island were the pelagic larvae of amphioxus which came from Taiwan Warm Current in the coastal waters of Fujian province. It is the first recorded species in Zhejiang Province, which will provide scientific data for the protection of amphioxus resources.