Non-invasive fecal samples have been widely used in monitoring animal sex hormone levels, but fecal treatment and hormone extraction need obvious species-specific manipulations in different species due to their different feeding habits and metabolism. In this study, we used wild and captive macaque (Macaca thibetana) to collect feces samples, and the contents of hormones were detected by using radiation immunoassay (RIA) after feces had been treated differently (wet feces，freeze-dried crushed feces，freeze-dried sifted feces) and the hormones had been extracted by various approaches (ethanol-heating method，methanol-ether method，ethanol-acetone method，methanol-ethanol method), in order to find optimal methods for detecting estradiol (E2) and progesterone (P4) levels in macaques under different feeding conditions. The data were analyzed using Two-Simple-Wilcoxon test. The results showed that the contents of estradiol and progesterone were the highest in feces treated by freeze-drying sifting method (Fig.1). There was significant difference in results between freeze-drying sifting method and freeze-drying crushing method in wild macaques (P < 0.01, Fig. 2), but there was no significant difference in captive macaques (P > 0.05，Fig. 2). The content of hormone in wet feces treatment was the lowest, which was significantly different from the other two treatment methods (P < 0.01, Fig. 1)；the content of E2 and P4 extracted by ethanol-heating method was the highest（Table 1）, which was significantly different from that of methanol-ether method (P < 0.01, Table 3), ethanol-acetone method (P < 0.01, Table 1) or methanol-ethanol method (P < 0.01, Table 1); Considering the average content of hormones, the simplicity of treatment process and the feasibility of extraction steps, it is recommended to use freeze-drying sifting method for the treatment of wild macaque feces. As the freeze-drying crushing method is easy to operate and there is no significant difference between the two methods, the freeze-drying crushing method is recommended for captive macaque feces. Ethanol-heating method is recommended for the extraction of fecal hormones from wild and captive macaque.