Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time-consuming and labor-intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential genecan(CRISPRi::CA) that encodes a carbonic anhydrase for CO(2)uptake. CRISPRi::CA comprises a whole-cell biosensor to monitor CO(2)concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation & Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony-forming units. Furthermore, the enzymatic performance of 5-aminolevulinic acid synthetase (ALAS), which converts glycine and succinate-CoA to release a molecule of CO2, has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time-saving, and flexible technology for the screening and inspection of high-performance enzymes, which may accelerate protein engineering in the future.