This study aimed to evaluate longitudinal changes in retinal blood flow in response to flicker stimulation and systemic hyperoxia in mice using a laser speckle flowgraphy (LSFG-Micro). The retinal blood flow in vascular area surrounding the optic nerve head was measured in 8-week-old male mice every 2 weeks until age 20-week. The coefficient of variation of retinal blood flow under resting condition was analyzed every 2 weeks to validate the consistency of the measurement. On day 1 of the experiment, retinal blood flow was assessed every 20 s for 6 min during and after 3 min flicker light (12 Hz) stimulation; on day 2, retinal blood flow was measured every minute for 20 min during and after 10 min systemic hyperoxia; and on day 3, electroretinography (ERG) was performed. Body weight, systemic blood pressure, and ocular perfusion pressure increased significantly with age, but the resting retinal blood flow and ERG parameters remained unchanged. Retinal blood flow significantly increased with flicker stimulation and decreased with systemic hyperoxia, independent of age. The LSFG-Micro provides consistent and reproducible retinal blood flow measurement in adult mice. Longitudinal assessments of retinal blood flow in response to flicker stimulation and systemic hyperoxia may be useful indexes for noninvasive monitoring of vascular function in retinas.