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[Display omitted] • First report on spectrofluorimetric determination of nitroxynil in its pure form and VET formulations. • Determination of fluorescence quantum yield of nitroxynil using single-point method. • Detection of nitroxynil residues in bovine meat, liver, kidney and milk samples. Because of the frequent treatment of dairy cows with nitroxynil (NTX), the presence of the later residues in animal tissues and milk has a significant concern. The quantum yield of the reaction product was calculated. A highly sensitive and rapid spectrofluorometric method for determining the anthelmintic drug (NTX) residual amounts is developed. The proposed approach is based on using Zn/HCl to reduce NTX nitro group to an amino group, resulting in a highly fluorescent derivative that was detected at each of λ em 302 nm and 364 nm after excitation at λ ex = 277 nm. The experimental conditions were carefully studied and optimized. The proposed approach showed good linearity (r2 ≥ 0.9998) with linearity range of 10.0–100.0 ng/mL with a detection limit of 1.89 ng/mL, 1.27 ng/mL and quantification limit of 5.73 ng/mL, 3.86 ng/mL at λ em 302 nm and 364 nm, respectively; that is far below the Minimum Regulatory Limits (MRLs) of NTX in animal tissues and milk. The proposed approach was successfully applied for the analysis of the drug in its veterinary formulations, and the obtained results agreed well with those of the official British Pharmacopeia method. Moreover, the proposed method's application was extended to efficiently determine the residues of nitroxynil in meat, liver, kidney and milk samples. [ABSTRACT FROM AUTHOR]