Clown loach Chromobotia macracanthus is one of Indonesian native species and as a key species for ornamental aquaculture. The problem in mass production of this species are the low of growth rate which causes a long period of rearing and low of survival rate. The purpose of the present study was to evaluate free glutamine supplementation at different doses in diet to increase physiological response, growth performance and survival rate of clown loach. Expe-rimental diets contained four different free glutamine levels, viz 0, 1, 2 and 3%. These diets were given to six repli-cate groups of 50 juvenile clown loaches. The fish were reared in each aquarium with dimensions of 40×30×30 cm3 for 60 days. Fish were fed four times a day at satiation. Parameters observed including intestinal glutamine concen-tration, villous and intestinal morphometry, intestinal protease activity, feed efficiency, nutrient retention, growth performance, survival rate, superoxide dismutase (SOD) activity, and malondialdehyde (MDA). The results showed that supplementation of 1% free glutamine significantly affected the morphometry of villi. The length and surface area of villi at a dose of 1% showed the highest values i.e., 320.44 ± 10.39 μm and 27,046.79 ± 250.54 μm2, respect-ively. The 1% dose also had a significant effect on protease activity (13.57 ± 1.92 mg units of protein-1) compared to the 0% dose. The 2% dose showed the highest SOD activity (0.82 ± 0.07 mg protein-1 unit) and the lowest MDA level was found at a dose of 3% (0.25 ± 0.02 nmol mg protein-1). Feed consumption with the supplementation of free glutamine has a significant effect on survival rate with the highest value reached 97.00 ± 1.00%, but no significant effect on intestinal morphometry, feed efficiency, nutrient retention and growth performance. Dietary with the sup-plementation of free glutamine is not able to improve growth performance, but can improve the physiological res-ponse and survival rate.