Abstract: In order to reduce the problems of low recovery rate and poor quality of sperm RNA in sheep semen caused by other somatic cell contamination and insufficient cracking, fresh Merino sheep semen was used as the research object in the experiment, and sheep semen was purified by Percoll+hSOF and Percoll+PBS density gradient centrifugation and PBS direct washing method, and then sheep sperm RNA was extracted by TRIzol（65 ℃） method and TRIzol（65 ℃）+ kit method.After the concentration, purity and integrity of RNA were detected, the RNA was reverse-transcribed into cDNA, and the PRM1 gene（encoding protamine 1） and CDH1 gene（encoding epithelial cell cadherin） were amplified by PCR to verify whether the extracted RNA was contaminated by somatic cells. The results showed that Percoll density gradient method could better move the somatic cells from sheep semen than PBS direct washing method, but the sperm recovery rate was low. The purity of RNA extracted by Percoll+PBS density gradient centrifugation was higher than what was extracted by PBS direct washing method, but the concentration was lower. The concentration of sperm RNA extracted by TRIzol（65 ℃） method was higher than what was extracted by TRIzol（65 ℃） + kit method, but the purity was lower.PRM1 gene was detected in the sperm RNA obtained by the two semen purification methods（Percoll+PBS density gradient centrifugation and PBS direct washing method） combined with the two sperm RNA extraction methods（TRIzol 65 ℃ method and TRIzol65 ℃+ kit method）, but CDH1 gene was not detected. The results indicated that the sperm RNA extracted by Percoll density gradient centrifugation combined with TRIzol（65℃） + kit method had a good effect, with no somatic cell contamination, and higher purity.