<p>This study aims to explore the potential of non-destructive, sensitive and advance confocal micro-Raman spectroscopy to analyze the effect of varying concentrations of iron oxide nanoparticles on physiological and biochemical responses of maize seedlings. The analysis of the acquired Raman spectra of the adaxial surface of the leaf of the control and iron oxide treated maize seedlings shows that nanoparticles affect the biochemical profile, growth and development of maize seedlings. The treatment of iron oxide nanoparticles increases the level of biochemicals like carotenoid, cellulose, carbohydrates, lignin, protein, pectin and aliphatic compounds present in the leaves of maize seedlings as compared to control in dose-dependent manner. Further the results of principal component analysis (PCA) demonstrate that the first three principal components explain the majority of variance in the Raman spectral dataset, with PC1 accounting for 73.25%, PC2 for 16.71%, and PC3 for 2.43%. This approach establishes confocal micro-Raman spectroscopy combined with chemometric analysis as a potential way of analyzing the interaction between nanoparticle and plants and provides insights into the beneficial role of iron oxide nanoparticles in enhancing biochemical constituents in maize seedlings.</p>