Background: The burden of stroke in China has increased dramatically in the past 30 years. Specifying the treatment according to the side of stroke in the brain might be an effective way to reduce the burden. Current imaging tools to identify the side of brain stroke, such as magnetic resonance imaging (MRI), are expensive and time-consuming. Hence, there is a great need for a rapid and inexpensive assessment. In this case, retinal image analysis is a possible approach for stroke side identification. This study aimed at determining the association between retinal characteristics and the stroke side and to establish a predictive model for further investigation.Methods: A total of 168 patients (89 left-sided stroke patients and 79 right-sided stroke patients) were recruited from the Shenzhen Traditional Chinese Medicine Hospital in the study. Retinal characteristics were analysed using an automated retinal image analysis (ARIA) system. Multivariable logistic regression was used to identify and develop predictive models.Results: Each unit increase in the right eye bifurcation coefficient of arterioles increased the risk of right-side stroke by 7.523 times (95% CI, 1.823–31.044). Additionally, an elevated bifurcation coefficient of venules in the right eye also increased the risk of stroke in the right side of the brain, with an odds ratio (OR) of 7.377 (95% CI, 1.771–30.724). A complex retinal composite score was also associated with a higher risk of right-side stroke (OR, 4.955; 95% CI, 3.061–8.022).Conclusions: This study demonstrated that retinal images can provide useful information for stroke side identification, and specific retinal characteristics may help predict the occurrence of stroke.Trial registration: Acupuncture Clinical Trail Redistry, ChiCTR1800019647, Registered 01 November 2018, AMCTR-OPC-18000228.