Probiotic fermented fruit extract is a new kind of food with potential health care efficacy. In this study, transformation of bioactive substances in Lactic acid bacteria (LAB) and yeast fermented kiwifruit extract were researched. The highest levels of total polyphenol, superoxide dismutase (SOD) were found in Lactobacillus paracasei LG0260 fermented kiwifruit extract with 2.31 ± 0.06 mgGAE/g, 369 ± 12.73 U.mL−1, respectively. LAB fermented kiwifruit extract dramatically had a highest vitamin C (VitC) concentration during the fermentation, while yeast and natural fermented samples were decline steadily. Another interesting founding, a highest level of γ-aminobutyric acid (GABA) was found in Saccharomyces cerevisiae J2861 fermented kiwifruit extract with 24.132 ± 1.01 μg/mL. Furthermore, the main organic acids in fresh kiwifruit and yeast fermented kiwifruit extract were citric acid and malic acid. However, lactic acid and citric acid were the main organic acids in LAB fermented kiwifruit extract. A total of 43 kinds of flavor compounds in fresh kiwifruit and 88 kinds of flavor compounds in fermented kiwifruit extract were identified. Esters and alcohols in fermented kiwifruit system increased by the fermentation of selected bacteria which improved the taste and flavor. •Probiotic fermented kiwifruit extract is a new kind of health care efficacy food.•The evolution of biochemical components in long-term fermented system was proved.•The change of organic acids in fermented kiwifruit extract was revealed.•The flavor compounds of different fermented kiwifruit extracts were compared.