The influence of sub-temperature quenching temperature on the strength and hardness of 65Mn steel during austenite inverse phase transformation is studied and the microstructure and property are analyzed. The results showed that in the range of 760~810°C , the strength and hardness of the 65Mn steel second sub-temperature quenched increased with quenching temperature increasing, reached the highest strength and hardness at 790°C quenching and then began to decrease. A small number of ferrite and ferrite dual-phase structure existed in martensite when quenching temperature was low. Fine martensite was obtained by second sub-temperature quenching due to the smaller austenitic crystal grain and austenitic nucleation sites. The mechanical properties of samples second sub-temperature quenched are better than that of conventional samples 830°C once quenched.