This study aimed to develop an algorithm to determine the optimal injection position in olive flounder, Paralichthys olivaceus when a vaccine is injected into the fish by using a vision‐based automatic equipment measruing the total length (TL), width and weight of the fish. Over a 5‐month period, 500 olive flounders were examined to analyze the relation of the fish size and to the shape and location of its abdomen, using radiography, a ruler plate and scale. There were significant correlations between the TL and the shape and location of the abdomen. The abdomen was located 0.232TL – 2.7221 mm (R2 = 0.8787) from the end of the mouth of the fish. The height and width of the abdomen in the fish were 0.1292TL + 1.8768 mm (R 2 = 0.7935) and 0.183TL–5.9791 mm (R 2 = 0.8641), respectively. The injection point in the abdominal region avoiding organs was determined by calculating the center of gravity of the abdomen volume. This can be expressed as g (0.2759TL – 2.0965, 0.1295TL – 4.2325) on the basis of TL line coordinates. The injection point by the expressed coordinates had errors for the x and y axes as 12.15 mm and 8.28 mm, respectively. These were enough to use the algorithm to injection for the equipment. This automated method to determine the position of any part in the fish can also be used for other purposes, for example, intramuscular injection or auto ‐tagging of fish.