The invention relates to the technical field of biological information, and discloses a method for noninvasive identification of pseudo-female Chinese softshell turtle through fluorescent quantitativePCR. The method comprises the following specific steps of setting a pair of primer groups for sexual identification of the pseudo-female Chinese softshell turtle, wherein the primer groups comprise primer pairs designed by DNA sequences of target genes 18S rRNA and Gapdh; performing fluorescent quantitative PCR data processing by adopting a 2-[delta][delta]Ct method, using the Gapdh as a reference gene, and calculating the relative copy number of the 18S rRNA in a genome; and by utilizing the fact that the DNA sequence copy number of the 18S rRNA on the W chromosome of the Chinese softshell turtle is higher than that on the Z chromosome, screening out the pseudo-female Chinese softshell turtle with the ovarian gonad and the ZZ genotype. According to the identification method, the fluorescent quantitative PCR technology is used for sexual identification of the Chinese softshell turtle, detection can be achieved only through a trace amount of blood, non-invasive collection of tail bloodof the Chinese softshell turtle has no influence on health of the Chinese softshell turtle, the method has the characteristics of being efficient, high in sensitivity, good in stability and high in specificity, the blank of pseudo-female Chinese softshell turtle identification is filled, breeding of all male softshell turtle fries is facilitated, and the breeding benefit is improved.