The invention discloses a method for separating and measuring moxifloxacin hydrochloride and an enantiomer thereof. According to the method, high performance liquid chromatography is adopted, a reversed phase column is used, a chiral reagent serves as a mobile phase, the enantiomer in the moxifloxacin hydrochloride is positioned according to a moxifloxacin hydrochloride racemate, the enantiomer of the moxifloxacin hydrochloride existing in various dosage forms including sustained release type, controlled release type or common tablets, capsules, granules, oral liquid, injection, eye drops, nasal drops, auristilla, suppository or injection can be checked, the quality standard of the conventional moxifloxacin hydrochloride is improved, and a basis is provided for setting an enantiomer measurement standard of the moxifloxacin hydrochloride in the future.