The invention relates to a Nile tilapia sex chromosome DNA (deoxyribonucleic acid) library construction method, belonging to the technical field of molecular biology. The method comprises the following steps: Nile tilapia head-kidney cell culture, cell hypotonic treatment, cell fixation, tabletting, chromosome cutting, separation, enzyme digestion and joint connection, amplification and recovery, and segment sequencing and analysis. The method is standardized, and is simple to operate. The proportion of lymphocytes in the chromosome separation phase prepared from the head-kidney cells is higher than that of hemocytes, thereby ensuring the favorable chromosome separation phase in the same visual field, and obviously enhancing the tabletting efficiency. By using the method, the neutral chromosome can be obviously distinguished from other chromosomes in the high power field. The method has the advantages of high cutting stability and high repetitiveness, lowers the separation difficulty of the Nile tilapia sex chromosome, and enhances the library construction specificity.