Otolith microstructural features of larval and juvenile fish, and especially the confirmation of the first annulus, are important for studying the early life history of fish. In this study, otolith microstructure analysis was performed on 318 young Gymnocypris selincuoensis (99 larvae and 219 juveniles) collected from Lake Siling Co from May 2010 to June 2011. The time of the first annulus formation of otoliths in juveniles was confirmed. The results showed that lapilli were the most suitable otoliths for age determination of G. selincuoensis among the three pairs of otoliths. By comparing the microstructure features of the otolith edge of the wild juveniles before and after overwintering, the first annulus formation time of the lapilli of the 2010 generation of juveniles was confirmed to be from April 17 to May 3, 2011 using the daily increment analysis. On the lapillus, an opaque band was formed from mid-April through early November (the growing season of G. selincuoensis), and a translucent band (i.e., the annulus) was formed from early November to mid-April of the following year (the slow growth stage). The first daily increment outside of the first annulus was formed at an average water temperature of 1.69 degrees C, which is the lowest reported water temperature at which fish were able to form daily increments. Our proposed method to determine the time of the first annulus formation through otolith daily increment analysis provides a referential way for confirming annulus formation in fish. The high temporal resolution (at day level) of this method in estimating annulus formation time highlights its application potentials in studies of growing season length estimation and fish response to water temperature change, which can assist with fish population management and conservation challenges arising from climate and environmental changes.