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 gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO2 uptake. CRISPRi::CA comprises a whole‐cell biosensor to monitor CO2 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.