Interferons (IFNs) play a significant role in the immune regulation of vertebrates, and three types (IFN-I, IFN-II, and IFN-IV) have been identified in bony fish. This study investigated the IFN genes in Acipenser sinensis, Acipenser ruthenus, and Acipenser oxyrinchus from the Acipenseridae, as well as in Polyodon spathula from the Polyodontidae. Using a series of bioinformatics methods, three IFN-I subgroups were identified: IFNb, IFNe, and IFNf. A conserved structure of scn4a/IFN-I/arhgap27 was found in the IFN-I gene cluster, and the IFN-I genes were tandemly arranged on the chromosomes in a 4C-2C pattern. Notably, unlike other bony fish, the sturgeon IFNf lacked the third cysteine residue, allowing for the formation of only one S-S bridge. Furthermore, IFN-II (IFNγ) and IFN-IV (IFNυ) were also identified in sturgeon, with IFN-IV being identified for the first time in sturgeon. Phylogenetic tree and sequence analyses revealed a high degree of homology among IFN genes across different families within the Acipenseriformes, suggesting that these genes have remained evolutionarily conserved. The systematic identification of sturgeon IFNs has provided resources for future studies on the antiviral immune response in sturgeon.