The invention discloses a spin-labeling rotenone oxime ester compound, its preparation method and application. The structural formula of the spin-labeling rotenone oxime ester compound is as show in the formula, wherein the substituent R can be 2,2,5,5-tetramethyl pyrroline nitronyl nitroxide radical, or 2,2,5,5-tetramethyl tetralin pyrroline nitronyl nitroxide radical, or -2,2,6,6-tetramethyl-tetralin pyridine nitronyl nitroxide radical or 2,2,6,6-tetramethyl-piperidine nitronyl nitroxide radical. The spin-labeling rotenone oxime ester compound provided by the invention can be applied in the preparation of antitumor drugs.