The use of pesticides in agriculture can make their way into the earth and wash into the amphibian system causing ecological stress. This study aims to understand the changes occurring in gill tissues as a result of fenvalerate exposure using Fourier-transform infrared spectroscopy. The intensity ratio of the selected bands I/I I/I, and I/I measures changes in proteins, lipids, and carbohydrates. Curve-fitting analysis was performed in the selected band region to analyze the quantitative changes of proteins, lipids, and carbohydrates. The band area ratio of CH/asCH+ sCH shows the absence of a long chain of fatty acids due to fenvalerate treatment. The band area ratio of asCH/sCH increases for higher sublethal concentrations, which shows the lower disorder of lipid acyl chain flexibility. A decrease in lipids was found in lower sublethal concentrations. The secondary structure of proteins affirms β sheet development. Carbohydrate metabolism of gill tissues demonstrates a decrease in glycogen contents. A further decrease in glycogen content and an increase in lactic acid were observed when presented to a fenvalerate concentration. PCA plots indicate distinct variations among the biochemical parameters of the gill tissues. This study provides a quantitative examination of assessing pesticide toxicity in aquatic environments.