Objective To subject vaginal smears of female SD rats and C57BL/6J mice to rapid Gram staining, observe the cell morphology, and develop a simple staining method to rapidly identify the estrous cycle stage. Methods Vaginal smears from 12 female SD rats and 12 female C57BL/6J mice at 56~62 days of age were obtained by the vaginal swab method every day at 9:00, dried, and then subjected to rapid Gram staining for continuous observation over 12 days. Cell morphology was observed under a light microscope to determine the estrous cycle stage. Results Rapid Gram staining was performed quickly and simply, bright in the observation field with a clean background, and provided a complete overview of the changes in the estrous cycle of rats and mice. Conclusions The rapid Gram staining method can easily and intuitively judge the estrous cycle stage of rats and mice, staining steps can be optimized, abnormal conditions in the staining can be analyzed stepwise, and the staining time of each smear can be shortened to less than 1 min. The method is easy for novice experimenters to master and can be applied widely.