The AT-hook motif nuclear localized (AHL) gene family is a highly conserved transcription factors involved in plant growth, development, and stress responses. To reveal the basic characteristics of AHL family in spinach, the members of the spinach SoAHL family were identified at the whole genome level, and their physicochemical properties, gene structure, conserved motifs, promoter elements, and salicylic acid-responsive expression profiles were analyzed in this study. Resulted showed that there were 19 SoAHL family members in spinach genome, which were unevenly distributed across six chromosomes. These SoAHL members can be classified into three branches, with 10 members in subfamily I and 9 members in subfamily II. Sequence composition of PPC and AT-hook conserved motifs varies among subfamilies; most of the SoAHL genes were located in nucleus, cytoplasm, and mitochondrion. Members of subfamilies I of SoAHL have no introns, while members of subfamily II contain 4–5 introns. The varying numbers of cis-acting elements about hormones and abiotic stress responses were distributed upstream of the promoters of SoAHL members. The SoAHL genes can be expressed in roots, leaves, and petioles, with most genes expressed at higher levels in roots. The expression of two SoAHL genes (SOV6g041850.1 and SOV2g038950.1) were significantly induced by salicylic acid treatment. The expression profiles and salicylic acid-induced expression levels of SOV2g031340.1 and SOV4g018880.1 were highly correlated with folic acid content, which may play a role in spinach response to salicylic acid signalling pathway. The transient overexpression of SOV4g018880.1 increased the folate contents of spinach leaves by 1.27 times. Our results will lay the foundation for further resolving the function of spinach AT-hook genes.