Objective To investigate the relationship between vaginal intraepithelial neoplasia (VaIN) and human papillomavirus (HPV) infection, liquid-based cytology, cervical lesions and vaginal microecology, so as to provide reference for clinical diagnosis. Methods The clinical data of 145 VaIN patients admitted to Shanghai Songjiang District Central Hospital from June 2019 to June 2021 were retrospectively analyzed, and they were divided into the low-grade lesion group (74 cases) and the high-grade lesion group (71 cases) according to the severity of the disease. All patients were examined for HPV infection, liquid-based cytology, cervical lesions, and vaginal microecology, and the relationship between VaIN and liquid-based cytology, HPV infection, cervical lesions and vaginal microecology were analyzed respectively. Results The proportion of patients with low-grade squamous intraepithelial lesion (LSIL) and low-grade cervical lesions in the low-grade lesion group were significantly higher than those in the high-grade lesion group; the proportion of patients with high-grade squamous intraepithelial lesion (HSIL), positive high-risk HPV (HR-HPV) infection, multiple HR-HPV infection, high-grade cervical lesions, diversity of bacterial,increased, reduction of dominant bacteria, Nugent score > 3 points, acetylglucosaminidase positive, leukocyte esterase positive in the low-grade lesion group were significantly lower than those in the high-grade lesion group (all P<0.05); the results of Pearson correlation analysis showed that, VaIN was positively associated with the diagnostic severity of TCT, positive HR-HPV infection, multiple HR-HPV infections, severity of cervical lesions, increased diversity of flora, decreased dominant bacteria, Nugent score > 3 points, acetylglucosaminidase positive and leukocyte esterase positive (r =0.453, 0.436, 0.484, 0.389, 0.413, 0.385, 0.374, 0.365, 0.324, all P<0.05). Conclusion The severity of VaIN can be assessed by HPV infection, liquid-based cytology, cervical lesions and vaginal microecological detection, and then provide reference value for clinical treatment.