The invention provides a method for separating X sperms and Y sperms, belongs to the field of 
livestock breeding. The method comprises the following steps: diluting a latex 
stock solution, adding a 
buffer solution and a pancreatin solution, uniformly mixing, carrying out water bath overnight, centrifuging to remove supernatant, and adding the 
buffer solution to obtain a latex suspension; and diluting an NT-4 
antibody, carrying out water bath, dropwise adding the NT-4 
antibody into the latex suspension, carrying out water bath, and placing at 
room temperature to obtain an 
antibody latex solution, and taking 
semen of a male 
mammal, adding a 
semen diluent, uniformly mixing, adding the antibody latex solution, uniformly mixing, and carrying out water bath incubation, wherein the upper half-layer solution in the incubation product contains Y sperms, and the 
lower half-layer solution in the incubation product contains X sperms. According to the invention, the method does not generate 
side effect, does not affect 
maternal health, does not reduce 
semen quality, reduces the malformation rate, is stable in separation effect and suitable for wide popularization and application, is simple and convenient to operate, improves the 
sperm separation efficiency, and can greatly reduce the 
workload of operators, save manpower, and reduce the labor cost.