This was a study of the fibrinolytic activities of oven dried and freeze-dried basidiocarps of Auricularia polytricha. Assessment of the effect of processing methods on fibrinolytic activity was by using both a fibrin plate assay and the Folin-spectrophotometric method. The aqueous two-phase system (ATPS) was used to further concentrate and recover fibrinolytic crude extract. The evaluation of system parameters such as the polyethylene glycol (PEG) molecular weight, concentration of PEG, as well as salt, concentration of crude and system pH was done in order to optimise the recovery of the fibrinolytic activity of A. polytricha. The fibrinolytic enzyme was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). In these studies, the crude extract and partially purified crude extract of freeze-dried A. polytricha basidiocarps showed positive fibrinolytic activities in fibrin plate assay. Extracts of freeze-dried A. polytricha basidiocarps had higher fibrinolytic activity, 10.83 U/mg, compared to oven-dried, 5.46 U/mg. PEG 8000-phosphate ATPS was found to be suitable for the primary recovery of fibrinolytic enzyme/s from the extract. Hence, the ATPS comprising a volume ratio equal to 1.5, PEG 8000 20.0% (w/w), phosphate 11.6% (w/w), system pH 7.0 loaded with 25% (w/w) of crude extract from freeze-dried A. polytricha sporocarps was found to be suitable with a purification fold of 7.01-fold (with 78% recovery of enzyme activity). The molecular weight of the enzyme was 66 kDa based on SDS-PAGE. (c) 2013 Elsevier B.V. All rights reserved.