This study investigated the effects of incorporating Salep, Orchis mascula into the diet of zebrafish (Danio rerio) on physiological indices and stress responses following transport stress. A total of 288 zebrafish with an average weight of 0.23 ± 0.01 g, were fed four experimental diets for 8 weeks: a control diet (C, basal diet without Salep) and three Salep-supplemented diets, including OM0.5 (0.5%), OM1 (1%), and OM2 (2%). At the end of the experiment, fish were subjected to transport stress two one hour, and biochemical indices, hepatic enzyme activities, and stress responses were evaluated. Results showed that diets containing 1% and 2% Salep significantly reduced plasma glucose, cortisol, cholesterol, lactate dehydrogenase, and aspartate aminotransferase activity (P < 0.05), while alkaline phosphatase activity peaked in the OM1 group (P < 0.05). However, triglyceride levels and alanine aminotransferase activity increased in the 2% group (P < 0.05). Based on these findings, using 1% Salep supplementation is recommended to enhance hepatic health, reduce stress, and regulate biochemical metabolism in zebrafish aquaculture.