A reliable QuEChERS-ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) analysis method was developed for the simultaneous determination of 13 steroid hormones(nrolone, androstenedione, methyltestosterone, testosterone, norethindrone, medroxyprogesterone, progesterone, diethylstilbestrol, hexan-stilbestrol, estradiol, estrotriol, cortisone, hydrocortisone) in Testis et Penis Cervi. The samples were extracted with methanol and purified by QuEChERS. Subsequently, the samples were separated by ACQUITY BEH C_(18) column and detected in the multiple reaction monitoring(MRM) mode under electrospray ionization in the positive and negative ion modes, respectively. Significant differences in the content of thirteen steroid hormones in Testis et Penis Cervi between the sika deer at different periods and the red deer were observed. The content of testosterone(10.88 μg·kg~(-1)) and hydrocortisone(12.82 μg·kg~(-1)) in Testis et Penis Cervi derived from rutting sika deer was significantly higher than the content of testosterone(1.05 μg·kg~(-1)) and hydrocortisone(0.73 μg·kg~(-1)) from antler growth stage. The content of progesterone in Testis et Penis Cervi derived from red deer was 6.07 μg·kg~(-1), significantly higher than that from sika deer. The content of progesterone in the testicle of red deer reached 27.46 μg·kg~(-1), 4.5 times greater than that in the penis of red deer. The sensitivity, accuracy, and precision of the method can meet the detection requirements, and the developed method is suitable for the measurement of hormones in animal-derived food.