[Objectives] Wildlife feces have the advantage of being easy to collect, preserve and transport as a non-invasive sample and are widely used for physiological metabolite extraction and to explore the physiological status of wildlife, with fecal steroid hormones being the most commonly used indicator. There are few researches on the systematic comparison of each extraction step to optimize the extraction method, which may lead to uncertainty in the determination results. This study used captive Forest Musk Deer feces as experimental samples. A comparison of four steps, including fecal sample amount, grinding method, single or repeated extraction, and drying or non-drying, was set up to explore the influence of different factors on extraction contents and stability of results, so as to optimize the extraction method of steroid hormones in Forest Musk Deer feces. [Methods] Fecal progesterone and cortisol contents were detected by enzyme-linked immunoassay and radioimmunoassay respectively. Data were analyzed using one-way ANOVA or independent-samples t test. [Results] The results showed that there were no significant differences in progesterone and cortisol contents between the 0.1 g, 0.3 g and 0.5 g sample groups, but the stability of the hormone measurements was poor in the 0.1 g group (Fig. 1). The contents of progesterone extracted from feces ground by the grinder were significantly different from that ground with liquid nitrogen by a mortar and pestle (t = 3.793, df = 12, P < 0.05), but there were no significant differences in cortisol between the two groups (t = 0.307, df = 12, P > 0.05) (Fig. 2). There were significant differences in both progesterone and cortisol contents between single and repeated solvent extraction (t =﹣4.459, df = 13, P < 0.05; t =﹣16.077, df = 7.434, P < 0.05) (Fig. 3), and no significant differences in progesterone and cortisol between the supernatant (30% ethanol) drying and non-drying groups (t =﹣1.502, df = 13, P > 0.05; t = 0.600, df = 14, P > 0.05) (Fig. 4). [Conclusion] Therefore, the method of extracting steroid hormones from Forest Musk Deer feces is optimized to use feces ground by the grinder and more than 0.1 g, the supernatant should be repeated solvent extraction and no drying (under the premise of verifying that the concentration of ethanol in the liquid to be tested does not affect hormone determination), which could achieve accurate, fast and convenient effect.