<p>Creatinine clearance is used to reflect the glomerular filtration rate to assess kidney function. Creatinine degradation-related enzymes have been used for creatinine detection in clinical medicine. The mixture of spores encapsulating either creatininase or creatinase or sarcosine oxidase could mediate a three-step reaction to produce hydrogen peroxide from creatinine. In this study, to achieve consecutive and efficient creatinine detection, degradation enzymes creatininase, creatinase, and sarcosine oxidase were co-encapsulated in a single <i>Saccharomyces cerevisiae</i> spore. The co-encapsulation spores performed high specific activity and enzymatic properties and converted creatinine to H₂O₂, which was 160% higher than the mixture of spores that individually expressed these three enzymes. The detection condition of co-encapsulation was optimized for the store at room temperature and resistance to environmental stresses. The <i>S. cerevisiae</i> spores can co-encapsulate enzyme families and catalyze consecutive reactions in the spore wall, having potential application prospects.</p>