<p>Tissue culture is preferred for solving the poor efficiency of conventional propagation of date palm (<i>Phoenix dactylifera</i>), an economically important woody plant with multiple uses. This study investigated the impact of using 24-epibrassinolide (24-epiBR) and plant growth regulators on the growth attributes and some biochemical constituents of in vitro implanted ‘Barhee’ date palm. The combination of 1.0 µM 24-epiBR and 22.2 µM 6-benzylaminopurine (BAP) was the most effective in terms of response rate and the number of shoots per jar (87.50% and 12.07 shoots/jar). The addition of 24-epiBR and BAP to the culture medium was more effective in accumulating the content of total soluble carbohydrates, free amino acids and soluble protein, where the best results, 4.36, 1.88, and 1.87 mg g⁻¹, respectively, were obtained using 1.0 µM 24-epiBR and 22.2 µM BAP application. The above treatment also increased endogenous cytokinin content, compared with other treatments. Up to 93.39% of the shoots were rooted on the 1/2 Murashige and Skoog (MS) medium with 1.0 µM 24-epiBR and 2.68 µM naphthalene acetic acid (NAA) and grew well in outdoor soil. Furthermore, the combined use of the above formulation resulted in the highest content of endogenous indole-3-acetic acid (IAA) (2.082 μg g⁻¹), compared with other treatments. No phenotypic differences were observed among the regenerated plants. This observation was validated using polymorphic RAPD and ISSR markers. The results may indicate that the micropropagation protocol in this study, induced by 24-epiBR in combination with plant growth regulators, effectively enhances the in vitro multiplication rate in <i>Phoenix dactylifera.</i></p>