ObjectiveVeterinary care of aquatic species, particularly fish, is limited by a lack of knowledge regarding their unique physiology. Tissue enzyme activities measured in plasma are used for assessing function and potential damage to specific organs and tracking disease progression in live animals. The objective of this study was to identify tissue(s) of origin and plasma concentrations for specific enzymes in healthy Golden Trevally Gnathanodon speciosus. We hypothesized that enzymes would exhibit tissue‐specific tropisms, with higher activities in one or more tissues compared to others.MethodsSix fish were randomly selected from a public aquarium population to obtain antemortem blood samples. The fish were then euthanized, and tissue samples were collected via gross necropsy. Six enzyme activities and two chemical analytes were examined across samples of plasma and 10 tissues from each fish.ResultEnzyme activities exhibited significant organ specificities. Aspartate aminotransferase, lactate dehydrogenase, and creatine kinase levels were highest in skeletal muscle, with variably high levels in gonads. Alkaline phosphatase levels were highest in the kidney, spleen, and liver. Alanine aminotransferase levels had high specificity for the liver. Gamma‐glutamyl transferase was only detectable in the kidney and plasma.ConclusionThis work establishes baseline tissue enzyme origins for Golden Trevally, which will aid clinicians in diagnostic interpretation of blood chemistries and improve veterinary care for this understudied fish species.