Humic acid enhances wheat growth by modulating auxin and cytokinin biosynthesis pathways. This study investigated the effects of humic acid (HA) on wheat seedling growth and gene expression related to auxin and cytokinin biosynthesis. The results showed that HA treatment significantly improved shoot and root growth in wheat seedlings. Gene expression analysis revealed that several genes involved in auxin and cytokinin biosynthesis were up-regulated in HA-treated seedlings compared to the control. Additionally, GUS expression analysis demonstrated that HA stimulates endogenous auxin and cytokinin-like activities. The study also showed that HA treatment increased root length, root surface area, root volume, and root hair length in wheat seedlings. The expression of genes involved in auxin biosynthesis, such as TAA1, YUC1, TAR2.1, GH3.1, GH3.2, and PIN9, was significantly up-regulated in HA-treated seedlings. Similarly, genes involved in cytokinin biosynthesis, such as IPT2, IPT5, LOG3, and LOG5, were also up-regulated. These findings suggest that HA improves wheat growth by modulating auxin and cytokinin biosynthesis pathways. The study is the first to demonstrate the biostimulant activity of humic acid using ARR5:GUS lines. The results indicate that HA enhances plant growth by activating endogenous phytohormone biosynthesis pathways. The study highlights the importance of understanding the molecular mechanisms underlying the growth-promoting effects of humic acid.Humic acid enhances wheat growth by modulating auxin and cytokinin biosynthesis pathways. This study investigated the effects of humic acid (HA) on wheat seedling growth and gene expression related to auxin and cytokinin biosynthesis. The results showed that HA treatment significantly improved shoot and root growth in wheat seedlings. Gene expression analysis revealed that several genes involved in auxin and cytokinin biosynthesis were up-regulated in HA-treated seedlings compared to the control. Additionally, GUS expression analysis demonstrated that HA stimulates endogenous auxin and cytokinin-like activities. The study also showed that HA treatment increased root length, root surface area, root volume, and root hair length in wheat seedlings. The expression of genes involved in auxin biosynthesis, such as TAA1, YUC1, TAR2.1, GH3.1, GH3.2, and PIN9, was significantly up-regulated in HA-treated seedlings. Similarly, genes involved in cytokinin biosynthesis, such as IPT2, IPT5, LOG3, and LOG5, were also up-regulated. These findings suggest that HA improves wheat growth by modulating auxin and cytokinin biosynthesis pathways. The study is the first to demonstrate the biostimulant activity of humic acid using ARR5:GUS lines. The results indicate that HA enhances plant growth by activating endogenous phytohormone biosynthesis pathways. The study highlights the importance of understanding the molecular mechanisms underlying the growth-promoting effects of humic acid.