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Organic solvents, such as methanol (MeOH) and ethanol (EtOH), are commonly used to extract and preserve steroids from animal excreta (feces, urine). As a result, the target substances are often in the solvent when they are subsequently analyzed using enzyme immunoassay (EIA). We examined steroid extraction from the feces of forest musk deer (Moschus berezovskii; collected in Sichuan Province, China, during 2017) using different concentrations of MeOH or EtOH (60%, 80%, 90%, or 100%). We also tested the effects of EtOH solvent on the accuracy of the EIA used to determine steroid concentration. Our results show that 100% MeOH and 90% EtOH resulted in the extraction of greater cortisol content from the feces of female forest musk deer compared with 90% MeOH and 100% EtOH (P < 0.05). Greater estradiol content was extracted with 100% and 90% EtOH compared with other EtOH concentrations or MeOH. The largest testosterone amount was extracted using 90% EtOH. In our evaluation of the effects of EtOH concentration on the accuracy of the EIA test, we found that inaccurate results appeared when the sample EtOH concentration was >7%. © 2020 The Wildlife Society. Noninvasive (feces, urine, etc.) methods to monitor the physiological state of animals are important, and both steroid hormone extraction and sample analysis are important components of this process, so it is important to choose suitable solvents for different target species. According to the results of this research, if ethanol is used as a solvent for the extraction of steroid hormones, the ethanol concentration should be diluted below 7% when using EIA, otherwise, ethanol may affect the results and lead to erroneous conclusions. [ABSTRACT FROM AUTHOR]