The invention discloses a separating method of optically-active pure isomer 21 (S)-Argatroban, which comprises the following steps: adopting 21(S) and 21(R) Argatroban as raw material; heating to reflux based on mixture of alcohol and water as solvent for 5-10h; cooling; stewing; filtering; obtaining white crystal product; drying; repeating 2-6 times; combining mother liquid; stripping solvent; detecting the content of 21 (S) through high-effect liquid-phase chromatography not less than 98%.