In this article, a simple, versatile, and mild method was used to fabricate porous enzyme microspheres using CaCO3 microparticles as templates. The preparation process involved three steps including coprecipitation of enzyme with CaCO3, cross-linking, and template removal. The preparation process was optimized and the obtained porous horseradish peroxidase (HRP) microspheres (p-HRP) were characterized. The p-HRP exhibited improved thermal stability and was more stable under alkali conditions than its free counterpart. These features are attractive for practical applications. Compared to free HRP, the K-m value of p-HRP was increased and the V-max value was decreased. The catalytic performance of p-HRP in removing phenolic compound and dye from aqueous solution was explored by using phenol and Direct Black-38 as model substrates. The results indicated that p-HRP exhibited high removal efficiency and moderately good reusability.