Repeated thermal treatment could impair the flavor of instant black tea (IBT) as the volatile compounds and characteristic constituents dramatically vary during the manufacturing process. Utilization of fresh leaves as raw material in IBT manufacture at low temperature may become an alternative to conventional extraction methods. This study aimed to improve the quality of IBT using fresh tea leaves by a two-stage enzymatic processing. The influence of combined enzymatic oxidation and extraction conditions for maximal extractable flavour (taste and aroma) characteristics from fresh tea leaves were investigated. The changes of aroma profiles and chemical constituents including theaflavins (TFs), thearubigins (TRs), theabrownines (TBs) and TRs/TFs ratio in converted tea brews under two-stage enzyme catalyzed fermentation and extraction conditions were further compared between different treatments, based on which the optimised conditions were determined. During the two-stage enzymatic processing, 75 kinds of volatile compounds were identified in all processed samples, and the contents of TFs, TRs, TBs and TRs/TFs ratio could be optimized by adjusting different procedures and parameters. The overall properties of final product were compared with two types of commercial IBT. High quality IBT with desired properties and antioxidant activity can be manufactured using fresh tea leaves by two-stage enzymatic processing approach.