The advancement of carbonic anhydrase (CA) activity and its inhibitor assays is crucial for drug development. Screening effective components from complex traditional Chinese medicine (TCM) systems as inhibitors is an important step in drug development. However, developing a simple and rapid method for this purpose remains challenging. Herein, a fluorescence (FL) sensing probe based on carbon quantum dots (CDs) integrated with a target affinity identification model for sensitive and specific CA detection and CA inhibitor screening was proposed. A FL sensing probe based on CDs was designed for the rapid detection of potential inhibitors in TCM samples on CA at first. Then the target affinity strategy was employed to accurately capture, isolate, and identify the active ingredient with inhibitory effects in the TCM sample. The developed method integrates two analytical models for screening CA inhibitors, significantly improving the accuracy and reproducibility of screening. The method was validated with positive, negative control and applied to screen CA inhibitors from 80 different TCM samples. The results revealed that ursolic acid and oleanolic acid from Chaenomeles speciosa (Sweet) Nakai, as well as cis-cinnamic acid and trans-cinnamic acid from Cinnamomum cassia Presl, are active ingredients that inhibit CA. Furthermore, isothermal titration calorimetry (ITC), molecular docking and dynamic simulations were employed to validate their interaction effects. Thus, this innovative detection method has great potential for rapidly screening CA inhibitor drugs. We believe that this strategy will stimulate further exploration and serve as a versatile and practical tool for screening enzyme inhibitors in TCM samples.