To investigate the situation of the ecological balance of vaginal flora of women with cervical intraepithelial neoplasia (CIN) III, and to study its correlation with the CIN recurrence after the operation of loop electrosurgical excisional procedure (LEEP) of the women. Methods: The clinical data of 200 women with CIN III and positive high-risk human papillomavirus (HR-HPV) infection who had received LEEP treatment in hospital from January 2018 to December 2023 were collected in study group retrospectively. 50 healthy women were selected in control group during the same period. The women in the two groups had received vaginal microecology detection. The related indexes of the vaginal microecology of the women in the study group were compared between before and after operation, and which of the women were compared between the two groups. According to the recurrence of CIN in 2 years after operation of LEEP or not, the women in the study group were divided into group A (women with the recurrence of CIN) and group B (women without the recurrence of CIN), and the postoperative vaginal microecological indicators and the other clinical data of the women were compared between group A and group B. The risk factors of the recurrence of CIN of the women after operation of LEEP were analyzed by Logistic regression analysis. Results: The proportions of the diversity Ⅱ-Ⅲ class of the preoperative vaginal flora intensity, the lactobacillus grade Ⅰ-Ⅱa, and the positive hydrogen peroxide (H2O2) of the women in the study group were significantly lower than those of the women in the control group. The vaginal pH value and the positive ratio of leukocyte esterase /sialidase (LE/SNA) of the women in the study group were significantly higher than those of the women in the control group. In the study group, the proportions of the vaginal flora density/diversity Ⅱ-Ⅲ class, the lactobacillus grade Ⅰ-Ⅱa, and the positive H2O2 of the women after operation had increased significantly, while the vaginal pH value and LE/SNA positive proportion of the women after operation had decreased significantly (all P<0.05). The recurrence rate of the women within 2 years after LEEP in the study group was 10.0%. The proportions of  the diversityⅡ-Ⅲ class of the preoperative vaginal flora intensity, the lactobacillus grade Ⅰ-Ⅱa, and the positive hydrogen peroxide (H2O2) of the women in group A were significantly lower than those of the women in group B. The vaginal pH, and the proportions of positive LE/SNA, menopause, preoperative multiple HPV infection, preoperative HR-HPV viral load ≥100 RLU/CO, invasion the vaginal wall of the lesion, positive lesion in surgical margin and postoperative persistent HR-HPV infection of the women in group A were significantly higher than those of the women in group B (all P<0.05). Logistic regression analysis showed that class Ⅱ-Ⅲ of the vaginal flora density and the grade Ⅰ-Ⅱa of the lactobacillus classification of the women were the protective factors of their CIN recurrence within 2 years after operation (P<0.05), but the higher pH value and the HR-HPV persistent infection of the women were the risk factors of their CIN recurrence (P<0.05). Conclusion: The vaginal flora ecology of the women with CIN III was unbalanced before operation of CIN by LEEP, and LEEP can effectively improve the vaginal flora ecology of the women. There is still a risk of recurrence of CIN after operation, and the CIN recurrence of the women is related to their vaginal flora density, lactobacillus grade and pH value, and their postoperative HR-HPV persistent infection.