A method for preparing chromosomes of red-spotted grouper adult fish belongs to the field of cell biology, and it includes pre-treatment of materials, preparation of kidney cell suspension, hypotonic treatment, fixation, dripping and staining; the pre-treatment of materials is Using the phytohemagglutinin injection method in vivo, inject PHA at the base of the pectoral fin of the experimental fish at a dose of 15 μg / g fish body weight, and inject colchicine solution at a dose of 2 μg / g fish body weight after 24 hours, and after 2.5-3 hours , take out the head kidney. The present invention adopts the local method of one-time injection of phytohemagglutinin, increases the injection concentration at the same time, can obtain good effect after 24 hours, and saves time. In the present invention, due to the characteristics of tissue cells of the red-spotted grouper, it is difficult to obtain complete cells when the hypotonic treatment is performed according to the prior art. Therefore, the present invention creatively changes the prior art and obtains a clear chromosome picture.