To establish an HPLC-electrochemical detection method to separate and determine teniposide and its related substances on a PhenomenexR LUNA Ph-Hex column, the condition of electrochemical detection was studied and the difference between electrochemical detection and UV detection was compared. Teniposide, lignan P, alpha-thenylidene lignan P(ATLP), picrothenylidene lignan P(PTLP) and the excipients of the injection were well separated using an acetonitrile-water（38:62） as mobile phase when the flow rate was 1.0mL/min, and the detection sensitivity was the highest when the working potential was set at＋0.7V using the glassy carbon disc electrode as the working electrode. Compared with the UV detection method, the sensitivity and selectivity of electrochemical detction are higher. It can be employed in the detection of impurities in Teniposide injection efficiently without any disturbance and the assay of teniposide.