An optimized karyote in-vitro separation kit comprises two reagents. The reagent 1 is composed of erythrocyte precipitant, sodium chloride or potassium chloride. The reagent 2 is composed of natrii diatrizoas and polysucrose No.400, natrii diatrizoas and polysucrose dextranum, or percoll, and has density of 1.076-1.090. An application method of the optimized karyote in-vitro separation kit includes directly adding 100ml of human marrow blood, umbilical cord blood or peripheral blood into 200ml of solution of the reagent 1, allowing for standing for 10-30 minutes, collecting supernate, centrifuging at 2000rpm (1100xg) for 5 minutes, discarding the supernate, adding remnant a centrifugal tube containing solution of the reagent 2, performing density gradient centrifugation for 30 minutes, controlling the rotation speed at 1500rpm, absorbing a karyote layer, performing centrifugal cleaning for once to three times, using 50ml of cell preservative solution for diluting, and taking 100 microliters for testing or self-testing.