In forensic practice, identifying the species of unknown bodily fluid stains can provide assistance in the qualitative analysis and investigation of cases, and vaginal fluid stains, as one of the common bodily fluid stains, are most commonly seen at the scene of sexual assault. At present, the commonly used vaginal peptidase or microscopic detection methods currently have drawbacks such as high false negative rates, poor sensitivity, and high requirements for sample integrity and background color. However, in forensic investigations, the test materials have specificity and scarcity, making it difficult to ensure their quantity and quality. Thus, in order to achieve rapid and sensitive detection of vaginal fluid stains, in this study, we combined nested PCR and isothermal amplification technology to construct a rapid detection system for suspicious vaginal fluid stains using lateral flow dipstick. This system achieves detection by detecting the specific marker microbial community Lactobacillus crispatus in vaginal fluid, and has a high sensitivity and accuracy, which can achieve detection at template quantities as low as 2.31 copies. More importantly, the system can achieve detection at a constant temperature of 37 °C without the need for complex instruments. It can provide rapid and sensitive identification results, providing assistance for subsequent forensic material extraction and individual identification.