Different concentrations of titanium oxide nanoparticles (TiO2NPs) have been frequently reported in treated wastewater used for the irrigation of crops. Luteolin is a susceptive anticancer flavonoid in many crops and rare medicinal plants that can be affected by exposure to TiO2NPs. This study investigates the potential transformation of pure luteolin in exposure to TiO2NP-containing water. In anin vitrosystem, three replicates of 5 mg L−1of pure luteolin were exposed to TiO2NPs (0, 25, 50, 100 ppm). After 48 h exposure, the samples were extensively analyzed by Raman spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, and dynamic light scattering (DLS). A positive correlation was found between TiO2NPs concentrations and the structural alteration of luteolin content, where over 20% of luteolin structure was allegedly altered in the presence of 100 ppm TiO2NPs. The increase of NPs diameter (∼70 nm) and dominant peaks in Raman spectra revealed that luteolin was adsorbed onto the TiO2NPs surface. Further, the second-order derivative analysis confirmed the transformation of luteolin upon exposure to TiO2NPs. This study provides fundamental insight into agricultural safety measures when exposed to air or water-borne TiO2NPs.