A novel organic-cobalt ternary macrocyclic compound HL-Co was constructed by cobalt ion with (N,N′)-5-(dibenzylamino)-N1(E),N′3(E)-bis(quinolin-2-ylmethylene)isophthalohydrazide, which was synthesized by introduction of quinoline and dansylamide groups into 5-aminoisophthalic acid. The recognition process of glutathione was studied with HL-Co by electrospray ionization mass spectrometry (ESI-MS) and ultraviolet spectroscopy (UV-vis). The results show that HL-Co forms a stable [3 3] macrocyclic structure in solution and can encapsulate glutathione at molar ratio of 1: 1. The absorbance at 320 nm was gradually increased when the glutathione was added to the solution of HL-Co, and the absorbance at 425 nm gradually decreased until the equilibrium. The isobestic point at 357 nm was obtained. An equilibrium constant log K(GSH) of 4.41 was calculated by fitting of Benesi-Hildebrand data. The minimum detection limit of glutathione was 1.0?10-5mol/L. It indicated that HL-Co has good recognition property on glutathione.