As a core component of vitamin B12, Co²⁺ is closely associated with the health, life performance, and productivity of ruminants. Therefore, the selective and sensitive detection of Co²⁺ is of great significance. In this work, using the Au–S bond as an “anchor”, a ternary nanocomposite (CDs-AuNPs-GSH) with synergistic functions was constructed by combining gold nanoparticles (AuNPs) with glutathione (GSH) through hydrogen bonding and electrostatic interactions with functional groups on the surface of carbon dots (CDs). The resulting nanocomposite was employed as a fluorescence sensor for Co²⁺ detection. Under optimal conditions at pH 6.0, the sensor exhibited a good linear relationship over the Co²⁺ concentration range of 0.5–125.0 mM, with a limit of detection (LOD) of 0.38 mM. The interaction mechanism between Co²⁺ and the composite was systematically investigated using various characterization methods. The results indicated that Co²⁺, owing to its strong coordination ability, enriches on the surface of the composite, subsequently triggering dynamic fluorescence quenching via an electron transfer pathway. The sensor was successfully applied to determine Co²⁺ in mixed livestock and poultry feed samples, with recovery rates ranging from 89.4% to 103.9%, demonstrating its potential for on-site detection in feed analysis.