Objective: To evaluate the effect of atropine penalization for treatment of moderate and severe anisometropic amblyopia. Methods: Retrospective cohort study was performed.Eighty moderate and severe anisometropic amblyopia pediatric patients aged 4-7 years old were enrolled from December 2011 to November 2017 in Tianjin Eye Hospital and Beijing Children's Hospital.The subjects were divided into atropine penalization group and patching group according to the treatment, with 25 cases of moderate amblyopia and 15 cases of severe amblyopia in each group.The best corrected visual acuity (BCVA) of amblyopia in atropine penalization group and patching group before treatment, 2 months, 4 months, 6 months and 8 months after treatment were compared.The binocular visual function of each group was compared before and after treatment.The compliance and complications were also recorded.This study adhered to the Declaration of Helsinki, and was approved by the Ethics Committee of Tianjin Eye Hospital (2016-42) and Beijing Children's Hospital (2019-k-33). Results: There were significant differences in BVCA between the two groups before and after treatment in cases with moderate amblyopia (Fgroup=3.104, P=0.032; Ftime=4.342, P=0.013); The BCVA of atropine penalization group was better than that of patching group at 2, 4, 6 and 8 months after treatment, the differences were statistically significant (all at P<0.05). There were significant differences in BCVA between the two groups before and after treatment in cases with severe amblyopia (Fgroup=3.149, P=0.016; Ftime=5.094, P=0.024); The BCVA of atropine penalization group was better than that of patching group at 2, 4, 6 and 8 months after treatment, the differences were statistically significant (all at P<0.05). The effective rates of stereopsis reconstruction in atropine penalization group were significantly higher than those in patching group in both moderate and severe amblyopia cases, and the differences were statistically significant (both at P<0.05). The compliance rate of moderate amblyopia in atropine penalization group was 96% (24/25), which was significantly higher than 76% (19/25) in patching group, the difference was statistically significant (χ2=4.153, P=0.042). The compliance rate of severe amblyopia in atropine penalization group was 100% (15/15), which was also significantly higher than 67% (10/15) in patching group, the difference was statistically significant (χ2=6.000, P=0.014). During the follow-up period, there were 4 cases with photophobia in the atropine penalization group.No adverse reaction was observed in the patching group. Conclusions: Compared with patching, atropine penalization is an effective method in treatment of moderate and severe amblyopia, with the advantage in rapid improvement of visual acuity, stereopsis recovery and compliance of treatment.Atropine penalization is worthy for clinical application. Copyright © 2019 by the Chinese Medical Association.