[Objectives] The present study investigates the characteristics and microstructure of the eggshell and eggshell membrane of Alligator sinensis eggs, under various conditions of surface mucous adhesion, fertilization status, and incubation time, in order to provide insights to improve the artificial hatching success of A. sinensis eggs. [Methods] We collected 21 eggs from the National Nature Reserve of Chinese Alligator in Xuancheng City, Anhui Province, China. This collection comprised 12 freshly laid eggs (including 3 fertile eggs with surface mucus, 3 fertile eggs without surface mucus, 3 infertile eggs with surface mucus, and 3 infertile eggs without surface mucus) and 9 fertile eggs with surface mucus at different days of oviposition, including 3 eggs at 10 d, 3 eggs at 20 d and 3 eggs at 30 d. Characteristics and microstructure of the eggshells and eggshell membranes of A. sinensis eggs were analyzed using a scanning electron microscope (SEM), under different conditions of surface mucous adhesion, fertilization, and incubation time. Data analyses were performed using the statistical package IBM SPSS (version 22.0). The results of fiber diameter and pore ratio were analyzed by Student’s t-test. The normal distribution test and homogeneity of variance test of data were analyzed by Shapiro-Wilk test and Levene test, respectively. The results have been showed as mean ± standard deviation (SD). P < 0.05 were considered as statistically significant. [Results] Observations showed pore channels traversing the shell surface in approximate concentric circles, hierarchically arranged. Blockages in some pore channels, likely composed of shell surface mucus, were noted. Initially, the eggshell with surface mucus appeared smoother and flatter during early incubation, developing numerous cracks over time (Fig. 1). Surface mucus appeared to reduce water evaporation, thus decreasing the formation of pore channels and cracks. These channels and cracks, attributed to the corrosive effects of carbonic acid and external forces, increased in diameter on the inner shell surface over time (Fig. 2). Irregular mammillary cones were noted on the inner surface of the eggshell, with air spaces between their bases. No significant difference in the number or morphology of mammillary cones between fertile and infertile eggs was observed (Fig. 3). The ultrastructure parameters of the inner surface of freshly laid eggs showed a porosity of approximately 11.85%, increasing with incubation duration to 30.44% at 30 d (Table 1). A negative correlation was noted between the presence of mammillary cones on the inner eggshell surface and incubation duration. Reticular keratin fibers in the eggshell membranes appeared random and sparse, with bud-shaped protrusions aligned on the fibers (Fig. 5). There was no significant difference for the fiber diameter and pore ratio between fertile and infertile eggs (t = 0.67, P = 0.52; t = 1.28, P = 0.23). And the fiber diameter and pore ratio between eggs with surface mucus and without surface mucus were also showed no significant difference (t = 0.21, P = 0.84; t = 1.53, P = 0.16) (Table 2). The fiber diameter did not differ significantly between fertile eggs at 0 day and 10, 20, 30 days after oviposition (t = 0.57, P = 0.60; t = 0.22, P = 0.84; t = 0.59, P = 0.59, respectively) (Table 3). The fiber pore ratio of the fertilized egg increased slightly from 14.14 ± 2.87% to 17.70 ± 1.12% within 30 d of oviposition, but the difference was not significant (t = 2.00, P = 0.15) (Table 3). [Conclusion] During incubation, fertile eggs absorbed external water, leading to crack formation and enhancing egg permeability, which aided embryo development by maintaining humidity. The presence of mammillary cones indicated increased permeability. The loose structure of the inner surface facilitated embryo development. Furthermore, the fiber diameter of eggshell membrane demonstrated a partial correlation with incubation duration and fertilization status.