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The optimization of explant selection and adjustment of plant growth regulators (PGRs) ratio may enhance the efficiency of micro-propagation of <i>Gardenia jasminoides</i> Ellis. The findings of the study suggest that the shoot tip proved to be the optimal explant for regenerating adventitious buds, with an impressive regeneration rate of 77.78% and the average number of adventitious buds being 2.86. The ideal medium consisted of Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (6-BA) at a 2 mg L⁻¹, indoleacetic acid (IAA) at a 0.2 mg L⁻¹, kinetin (KT) at 0.15 mg L⁻¹, resulting in an outstanding regeneration rate of adventitious buds reaching up to 91.11%. For rooting purposes, the best medium was found be half-strength MS supplemented with indoleacetic acid (IAA) 0.5 mg L⁻¹, achieving an rate for adventitious roots amounting to as high as 97.78%. The culture plantlets ultimately thrived, achieving an impressive transplanting survival rate of 93.33%. The application of PGRs was also found to enhance the regeneration of adventitious buds by increasing the ratios of endogenous hormones ZR/IAA and GA₃/IAA. Additionally, it facilitated the differentiation of adventitious roots by elevating the ratios of endogenous hormones IAA/ZR, IAA/GA₃, and ABA/GA₃. Our study would provide a theoretical reference for the establishment of an efficient gardenia tissue culture system and the industrial production of gardenia.