Aspergillus is a large tannase-producing genus able to provide enzymes which hydrolyse condensed tannins in waste coir residues. In this work, a tannase-acyl hydrolase (TAH) obtained from Aspergillus sydowii (SIS 25) was extracted and purified by PEG/citrate aqueous two-phase system (ATPS), biochemically characterized and applied for detannification of green tea. A statistical design (23) was used to evaluate the influence of the variables: molar mass of PEG (1000–6000  g/mol), PEG concentration (20.0–24.0% w/w) and sodium citrate concentration (15–20% w/w) at pH 6.0. ATPS efficiency was confirmed by obtaining two major chromatographic peaks (by size exclusion chromatography on a Superdex-G75 at 215 nm) with a unique peak responsible for the hydrolysis of 44% of the phenolic compounds in green tea. This study is focused on the obtention of a microbial tannase purified by ATPS, with potential for debittering and clarifying fruit juices. •Aspergillus sydowii was able to produce tannase with biotechnological potential.•The optimum pH for tannase activity was observed between 5.0 and 6.0.•The results showed that, after hydrolysis, the sweet aftertaste and overall acceptability improved.•The removal of phenolic compounds in green tea by Tannase was achieved.•The results showed that the phenolic compounds of the green tea were significantly influenced by the amount of tannase added.