Oxyfluorfen (2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene) is a nitrophenyl ether herbicide. Phytocystatins are crucial plant proteins which regulate various physiological processes and are also responsible for maintaining protease-antiprotease balance within plants. Thus, the present article deciphers the interaction of oxyfluorfen with garlic phytocystatin (GPC) through various spectroscopic and calorimetric techniques. The cysteine proteinase inhibitory (CPI) assay was done to assess the inhibitory action of garlic phytocystatin in the presence of oxyfluorfen. The garlic phytocystatin loses its inhibitory activity in the presence of oxyfluorfen. The complex formation of garlic phytocystatin-oxyfluorfen was shown by UV absorption spectroscopy. The intrinsic fluorescence experiment affirmed the quenching of garlic phytocystatin in the presence of oxyfluorfen. The Stern-Volmer quenching constant and binding constant was obtained as 6.89 x103 M-1 and 9.72x103 M-1 respectively. Synchronous fluorescence showed the alteration in the microenvironment around tyrosine residues. 3D fluorescence suggested the perturbation in the polarity around aromatic residues. The isothermal titration experiment suggests that the interaction of oxyfluorfen with garlic phytocystatin is a thermodynamically favorable reaction. Secondary structure alteration of garlic phytocystatin in the presence of oxyfluorfen was studied by circular dichroism (CD). The CD result showed a reduction in the α-helical content of garlic phytocystatin on interaction with oxyfluorfen. Consequently, all these outcomes affirmed the formation of garlic phytocystatin-oxyfluorfen complex along with the structural and conformational alteration. This study identifies and signifies that the exposure of oxyfluorfen induces stress within the plant system.