The invention relates to a method for constructing a sugarcane brown rust resistance gene positioning genetic separation population, which comprises the following steps: using sugarcane varieties withdifferent sources and high sensitivity to brown rust as female parents, using sugarcane varieties with high resistance to brown rust and no Bru1 gene as male parents, preparing, combining and hybridizing to obtain F1 generation seeds, sowing, screening a true hybrid F1 generation, planting a single plant of the true hybrid F1 generation, inoculating brown rust germs, investigating the disease incidence condition, evaluating the resistance according to a 9-grade standard, analyzing the disease resistance inheritance of a population of the true hybrid F1 generation, and selecting a true hybridF1 generation population of which the resistance-to-infection separation ratio of the hybrid offspring genetic separation population is approximately equal to 3:1 as the sugarcane brown rust resistance gene positioning genetic separation population. According to the method, a set of method for systematically constructing the sugarcane brown rust resistance gene positioning genetic separation population is established for the first time, the program is simplified, the operability is strong, the resistance-to-infection separation ratio of the constructed true hybrid F1 population is approximately equal to 3:1, the disease resistance gene of the constructed true hybrid F1 population is controlled by a dominant single gene, and the population can be used for gene positing of brown rust resistance.