The SFK gene family is a family of non-receptor tyrosine kinases that function in apoptosis, phagocytosis, inflammation, and other immune processes in innate immunity. This study first analyzed the gene structure and evolutionary relationship of SFK gene family members of Apostichopus japonicus by bioinformatics methods to elucidate the function of the non-receptor tyrosine kinases (SFKs) gene family in response to pathogen stress. qRT-PCR analyzed the temporal and spatial expression patterns of SFK gene family members under pathogen stress. In the present study, seven members of the SFK gene family were identified in A. japonicus, which were divided into two subfamilies SrcA and SrcB, distributed unevenly on three chromosomes. The domain and motif analysis showed that six members had conserved SH3 domain, SH2 domain and tyrosine kinase core domain except SrcB-3. Among them, five motifs that make up the core domain of tyrosine kinase existed widely in all members, and different members of the SH3 domain and SH2 domain had the same motif composition, indicating that the AjSFK family was relatively conservative in evolution. The analysis of SFKs gene expression patterns in different tissues showed that SFKs were widely expressed in various tissues of A. japonicus, and had apparent tissue specificity with high expression in the intestines and tentacles. In addition, the expression levels of SrcB-1-3 significantly increased after 6 h of infection with Vibrio splendidus, and the expression levels of SrcA-1 and SrcB-2 significantly increased after 24 h of LPS stress in vitro. The study showed that SFKs of A. japonicus had a high immune response and played an important role in responding to pathogen stimulation. This study was the first systematic identification and functional analysis of the SFK gene family in A. japonicus, and provided an important theoretical basis for disease resistance.