The optimization of explant selection and adjustment of plant growth regulators (PGRs) ratio may enhance the efficiency of micro-propagation of Gardenia jasminoides 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.