Nanomaterial-based artificial enzyme mimetics have attracted increasing attention because of their robust stability, adjustable activity, and cost-effectiveness. In this study, we developed a simple and effective method for the synthesis of highly dispersed ultrafine PdCo alloys with peroxidase- and catalase-like activities. The aberration-corrected transmission electron microscopy analysis verified that the cyanogel precursor in the mesoporous silica nanospheres (MSNs) was converted to PdCo alloy in NH₃ at a high temperature. The PdCo alloy was homogenously distributed in MSNs as ultrafine and monodispersed particles. By selectively removing the Co species from the binary alloy through an acid-leaching approach, the role of each component in the enzyme-like mimetics was systematically studied. Using glutathione (GSH) as the model analyte, the potential application of PdCo@MSNs in GSH detection from complex cell media was confirmed via colorimetric assay. The ultrafine alloy size, double mimetic activities, and abundant loading space of PdCo@MSNs make them promising not only in clinical diagnosis but also in overcoming hypoxia-induced photodynamic therapy resistance in tumor treatment.