Objectives The aim of the study was to evaluate the performance of a folate receptor-mediated tumor detection (FRD) assay for detection of cervical high-grade lesions. Materials and Method A total of 1504 patients with abnormal cytology and/or positive human papillomavirus (HPV) testing during primary screening from November 2014 to August 2015 were enrolled. The patients were recruited from the Peking University People's Hospital and 12 other hospitals. Folate receptor-mediated tumor detection was applied in all the patients before colposcopy to compare the detection rate, sensitivity, specificity, positive predictive value, negative predictive value, and coincidence rate with HPV and cytology tests according to the pathologic diagnosis. Results In the total of 1504 patients, 503 patients were negative for intraepithelial lesion or malignancy, 440 patients were cervical intraepithelial neoplasia (CIN) 1, 254 patients were CIN 2, 257 patients were CIN 3, 46 patients were squamous cell carcinoma, and 4 patients were adenocarcinoma in situ. The sensitivity of FRD was 77.72%, which was less than cytology (80.39%) and HPV testing (95.54%). The specificity of FRD was 60.02%, which was greater than cytology (30.12%) and HPV testing (14.95%). The coincidence rate of FRD to the pathologic diagnosis (66.62%) was also significantly greater than atypical squamous cells of undetermined significance cytology and above (48.87%) and HPV testing (45.01%, p < .0001). The detection rate of FRD for all grades of lesions increased with the severity of lesions. Conclusions Folate receptor-mediated tumor detection has a slightly lower sensitivity and a higher specificity than cytology and HPV testing for detection of CIN 2+. Simplicity of FRD requires less professional skill. Folate receptor-mediated tumor detection could be a candidate test for cervical cancer screening especially in low- and middle-income countries. However, FRD still needs more clinical trial data to demonstrate its ability in general screening population.