With the development of biopharmacology, many therapeutic enzymes have been developed for treatment of various diseases, including metabolic diseases, thrombotic cardiovascular diseases and cancers. Most of the approved therapeutic enzymes are hydrolases, which are used to clean the toxic organic compounds and biomacromolecules in vivo, such as saccharides, lipids, proteins and their aggregates. Due to the high catalytic activity, affinity and specificity of enzyme towards substrate, enzyme therapy has a shorter time frame and fewer side reactions compared to other therapeutic approaches. However, there are several critical bottlenecks that limit the effectiveness of therapeutic enzymes, including immunogenicity, short circulation time, and lack of tissue specificity. Many approaches have been used to overcome these challenges. Several second generation therapeutic enzymes with significantly improved effectiveness have been developed using molecular engineering technologies such as glycan modification and pegylation. In addition, enzyme gene therapy becomes an emerging approach for treatment of diseases caused by enzymes deficiencies. Here, we reviewed the current enzyme-based therapeutics, and discussed its advantages, challenges and future perspectives.