The SQS_PSY family (Squalene/phytoene synthase family), with squalene synthase (SQS) and phytoene synthase (PSY) as core members, comprises enzymes that catalyze the head-to-head condensation of isoprenoid precursors. These enzymes play pivotal roles in mediating plant responses to both biotic and abiotic stresses; nevertheless, their specific functions in soybean defense against Phytophthora sojae infection remain elusive. In the present study, a comprehensive bioinformatics approach was utilized to identify 12 SQS_PSY family members in the soybean genome, followed by subsequent analyses of chromosomal distribution, phylogenetic relationships, gene structures, conserved motifs, and cis-acting regulatory elements within promoter regions. Notably, multiple cis-elements responsive to biotic and abiotic stresses were detected in the promoter regions of SQS_PSY genes in soybean, implying their potential involvement in stress-responsive pathways. To elucidate their roles in defense, resistant and susceptible soybean cultivars were inoculated with P. sojae, and RNA-seq was conducted on sampled tissues. Integrated with quantitative real-time PCR (qRT-PCR) validation, our findings demonstrated that GmSQS1 exhibited differential expression patterns between resistant and susceptible cultivars at multiple time points post-inoculation. Furthermore, the functional role of GmSQS1 in enhancing soybean resistance to P. sojae was confirmed using a transgenic hairy root system. Collectively, this study preliminarily validated the functions of the SQS_PSY genes in soybean and offers novel insights into their potential application for improving resistance against Phytophthora root rot.