The invention relates to a method for detecting the content of an etimicin sulfate raw material and preparation of the etimicin sulfate raw material and relevant substances. The method adopts precolumn derivatization, the derivatization condition comprises taking 4ml sample with appropriate concentration, adding 1ml O phthalic aldehyde (OPA) solution into the sample, diluting the mixed solution with isopropyl alcohol to 10ml, heating the mixed solution at 30-60 DEG C in water bath away from light, and obtaining derivatization solution. The novel method for detecting etimicin sulfate adopts a high efficiency liquid chromatograph, the chromatographic condition is that an octadecylsilane chemically bonded silica filler chromatographic column has particle size ranging from 4-10mum, column specification is 150-250mmX4.6-8.0mm; flowing phase is divided into organic phase and aqueous phase, wherein the volume rate of the organic phase to the aqueous phase is 55-75:45-25, the organic phase is methyl alcohol or acetonitrile, the aqueous phase is glacial acetic acid containing 0.01M-0.03M ion pair reagent sodium heptanesulfonate, potential of hydrogen (PH) range is adjusted by ammonia water or ammonium acetate; flow speed is 0.5-1.0ml/min; sample feeding amount is 10-20mul; a detector is a conventional ultraviolet-visible light detector, and detection wavelength is from 330-350nm. The novel method for detecting etimicin sulfate adopts the conventional detector and the high efficiency liquid chromatograph, can achieve on-line detection of the content of the etimicin sulfate, the preparation of the etimicin sulfate and the relevant substances, and is simple, convenient, feasible, accurate and reliable.