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This study attempted to characterize three major classes of digestive enzymes in arowana fish, a popular and highly valuable ornamental fish species using specific substrates and inhibitors in biochemical and SDS-PAGE electrophoresis methods. Acidic pepsin-like enzymes were detected in stomach region at optimum pH of 1.5–2.0 and its importance is shown by a 96.1% inhibition by pepstatin. Trypsin and chymotrypsin were also detected in both pancreas and intestine of this fish. Metalloproteases were shown in electrophoresis studies and further biochemical specific assays confirmed the presence of carboxypeptidases and leucine aminopeptidase. Overall, these alkaline proteases displayed highest activities at pH range of 9.5–10 in both intestine and pancreas with inhibition rate ranging from 30% to 70% by different specific inhibitors. The detection of these enzymes in major digestive organs of this fish reveals a common protein digestion pathway of large polypeptides breakdown by pepsin in stomach, followed by endopeptidase and exopeptidase activities to further degrade chain polypeptides to amino acids for adsorption. While the presence of lipases is expected due to carnivorous nature of this fish, the detection of amylase (0.63–0.73 U) in this study gives indication that there is a possibility for arowana to assimilate a wider range of nutrient as previously though. This means that development of practical dry feed for arowana farming is possible in near future. [Copyright &y& Elsevier]