Oral potentially malignant disorders (OPMDs) with epithelial dysplasia can progress to oral squamous cell carcinoma (OSCC). Toluidine blue (TB) has been used in oral screening with various methods. This study aimed to evaluate the sensitivity and specificity of TB-containing electrospun fibers in detecting OPMDs with epithelial dysplasia compared to histopathology. The concentrations of polyvinylpyrrolidone (PVP) and TB were optimized for fiber diameter and puncture strength using the design of experiments method with a central composite design. These optimized fibers were tested on 36 suspected OPMD lesions in 30 patients, followed by a biopsy for histopathological examination. The quadratic and two-factor interaction models described the relationship between TB and PVP concentrations and fiber characteristics. The TB-containing electrospun fibers had 77.3% sensitivity and 35.7% specificity in detecting OPMDs with epithelial dysplasia. The TB-containing electrospun fibers offer advantages such as easy application, cost-effectiveness, and enhanced sensitivity in identifying OPMDs with epithelial dysplasia. However, their diagnostic accuracy is insufficient as a standalone diagnostic method, and they should be considered an adjunctive tool for detecting dysplastic changes in the oral epithelium. Thailand Clinical Trials Registry: TCTR20240429002.