Early detection of premalignant cervical lesions is essential for improving cervical cancer outcomes; however, current screening methods frequently lack adequate sensitivity and specificity. This research introduces a diagnostic platform that integrates lectin-based biosensors with spectral and multivariate analysis. The biosensors are composed of gold nanoparticles (AuNPs) conjugated with Maackia amurensis (MAA) lectin, which selectively binds to α2,3-linked sialic acid. Validation was performed using cervical cancer cell lines (SiHa, HeLa, C33A), fibroblasts, and cervical scrapes, and specificity was verified by enzymatic removal of sialic acids. Spectral data were obtained using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and analyzed by principal component analysis (PCA). Application of PCA to the 1600–1350 cm−1 spectral region, using 99% confidence ellipses, enabled clear differentiation between samples negative and positive for intraepithelial lesions in a double-blind study of 58 patients. The MAA biosensors exhibited high sensitivity and specificity, comparable to established diagnostic methods. These results indicate that the combination of ATR-FTIR spectroscopy, MAA lectin-based biosensors, and chemometric analysis provides a robust and reliable approach for early detection of premalignant cervical lesions, with considerable potential to enhance patient outcomes.