Background: Rutin is one of the flavonoid compounds found in different plant species. Rutin has powerful antioxidant properties. The main target of this study is to elevate rutin content in gardenia (Gardenia jasmonides Ellis) calli cultures. Different concentrations of phenyl alanine (2, 3, 4, and 5 mg/l) were used as a precursor for enhancement of rutin accumulation. Moreover, and to elevate rutin content, salicylic acid (SA) was used at different concentrations (34.5, 69, 103.5, and 138 mg/l). Various biochemical analyses, i.e., determination of total phenols, total flavonoids, and antioxidant activity during three periods, after 2 weeks (the first harvest), after 4 weeks (the second harvest), and after 5 weeks (the third harvest) of culture, were performed. In addition, determination of phenols and flavonoids content by highperformance liquid chromatography (HPLC) were estimated of the three periods. Results: The results showed that the highest value of total phenols (9.53 ± 0.55 mg GAE/g DW of callus) was recorded with 5 mg/l phenylalanine (PA) at the third harvest. While the highest value of total flavonoids (0.358 ± 0.002 mg QE/g DW of callus) was achieved using 2 mg/l P.A. at the third harvest. By using HPLC, it was found that the maximum rutin content was recorded with 3 mg/l PA at the third harvest (429 μg/g DW).Conclusion: This study showed that rutin and cinnamic acids were the major compounds detected with all treatments and during the three harvest phases of gardenia calli cultures. As well as results declared that the decreasing of cinnamic acid was correlated with the increasing of rutin content, and the maximum rutin content was recorded with applying of 3 mg/l PA at the third harvest (429 μg/g DW).