The purpose of this study is to observe the effects of glyceraldehyde cross-linking on sclera biomechanical strength and experimental myopia. 50 three weeks aged guinea pigs were randomly divided into 5 groups: A, B, C, D, E. Each group had 10 guinea pigs. The right eye was set as the experimental eye, the left eye was used as control. Group A: 7 days mask; Group B: 21 days mask, plus physiological saline retrobulbar injection at mask day 1, 8, 15; Group C: 21 days mask, plus 0.05 mol/L glyceraldehyde retrobulbar injection at mask day 1, 8, 15; Group D: 21 days mask, plus 0.5 mol/L glyceraldehyde retrobulbar injection at mask day 1, 8, 15; Group E: normal control group. Several parameters of 7 guinea pigs of each group were measured before and after deprivation (mask day 7, 14, 21), including ocular axial length, refractive error, ultimate stress (σmax) (MPa), ultimate strain(εmax) (%) and 6% elastic modulus (MPa). The effects of glyceraldehyde on adjacent tissues of the rest 3 guinea pigs were detected by histopathological and immunohistochemical studies. Differences between experiment eyes and contralateral, eyes were compared with paired t test and correlative analysis. The experimental eyes appeared the increase of the the vitreous cavity length, the axial length and myopia after deprivation. In group A, group B and group C, the differences of the length of the vitreous cavity (group A (2.991 ± 0.078) mm, group B (2.961 ± 0.038) mm and group C (2.936 ± 0.021) mm), the axial length(group A (7.263 ± 0.133) mm, group B (7.732 ± 0.099) mm and group C (7.665 ± 0.055) mm) and refractive error (group A (-2.214 ± 2.881) D, group B (-4.525 ± 2.415) D and group C (-1.607 ± 0.866) D) between the experimental eye and the fellow eye was statistically significant (vitreous cavity = 3.234, 4.758, 7.608; Pvitreous cavity = 0.018, 0.002, 0.001; axial = 3.198,, 4.758, 7.608; Paxial = 0.019, 0.002, 0.000; refraction = -7.120, -4.020, -6.334; Prefraction = 0.000, 0.005, 0.001). In group D and group E, there is no difference between deprived eye and control eye about the length of the vitreous chamber as well as axial length (vitreous = 0.542, -0.646; Pvitreous cavity = 0.607, 0.539; axial = 0.542, -0.646; Paxial = 0.607, 0.539). The experimental eye ((-3.921 ± 0.874)D) and the fellow eye ((-3.321 ± 1.205)D) of group D, the difference of diopter was statistically significant (refraction = -3.154, Prefraction = 0.020). At the end of the experiment, the change of diopter of experimental eye of group B, C, D, E was significantly different (F = 61.249, P = 0.000). The difference of diopter change between group B ((8.800 ± 0.616) D), group C ( (7.236 ± 2.198) D), group D ( (6.271 ± 1.112) D) and the normal control group ((0.934 ± 0.158) D) was statistically significant (PB = 0.000, PC = 0.000, PD = 0.000). At the end of the experiment, the ultimate stress and 6% elastic modulus of group B experimental eye was (7.988 ± 3.677) MPa (P = 0.002) and (19.938 ± 4.871) MPa (P = 0.001), decreased 10.06% and 34.17% respectively. On the other hand the ultimate strain was (28.6 ± 3.6) % (P = 0.034), increased 19.17%. After the cross-linking treatment, the ultimate stress and 6% elastic modulus of group C experimental eye was (9.244 ± 0.806) MPa (P = 0.001) and (26.180 ± 4.388) MPa (P = 0.031) , decreased 23.13% and 13.34%, the ultimate strain was (26.2 ± 1.0) % (P = 0.016) , increased 12.93% separately. The ultimate stress of group D experimental eye was (12.476 ± 2.507) MPa (P = 0.580), decreased 5.50%, 6% elastic modulus was (30.446 ± 3.410) MPa (P = 0.314), increased 6.53%, ultimate strain was (23.8 ± 1.8) % (P = 0.253), decreased 4.42% respectively. Ultrastructure examination showed that, decreased scleral thickness with fibers lined up in order, without inflammatory cells Infiltration. Expressions of matrix metalloproteinases-2(MMP-2) mainly decrease in the episcleral tissue. The stroma of choroid, and the outer plexiform layer. Glyceraldehyde is a safe and effective cross-linking agent that could significantly enhance the sclera biomechanical strength. Glyceraldehyde cross-linking method could effectively control the development of myopia in animal model.