Black-necked Crane (Grus nigricollis) is a national first-class protected wildlife. Due to its harsh natural environment and small population size in the wild, the Black-necked Cranes have been bred in captivity in many zoos and protected areas for protection, however, in low reproduction rate. In order to improve its reproduction rate, we explored the physiological characteristics of Black-necked Crane in captivity during breeding period using non-invasive sampling methods and behavioral observations. The enzyme-linked immunity was used to measure the contents of sex hormones (estradiol, progesterone and testosterone). SPSS was used to conduct one-way ANOVA and multiple comparisons on the data. The significance level was set as P = 0.05. The homogeneity of sample mean variance was determined by Levene’s homogeneity of variance test, Dunnett''s T3 test was used for multiple comparisons of data from different sampling periods. The results showed that: The estradiol level of Black-necked Crane in the breeding period was significantly increased, which could be used as one of the important physiological indicators to effectively monitor the physiological status of females in the breeding period (Fig. 1); The testosterone level of males in the early stage of reproduction was significantly increased, which was significantly different from that in the non-breeding period and other stages of breeding period, indicating that testosterone could effectively monitor the reproductive physiological state of males and provide a theoretical basis for the selection of appropriate time for artificial extraction (Fig. 2 and 4); The progesterone level in the middle reproductive period of the females with successful mating was significantly higher than that in other stages, indicating that this indicator can effectively monitor the mating status of the females (Fig. 1). In conclusion, monitoring the appropriate physiological parameters can provide a theoretical basis for the breeding of captive Black-necked Crane and improve its breeding rate.