Membrane proteins are vital resources for developing biosensors. TMEM120A is a membrane protein associated with human pain transmission and lipid metabolism, and recent studies have demonstrated its ability to transport ions and bind to coenzyme A (COA-SH), indicating its potential to develop into a single-molecule sensor based on electrical methods. In this study, we investigated the ion transport properties of TMEM120A and its homolog TMEM120B on an artificial lipid bilayer using single-channel recording. The results demonstrate that both proteins can fuse into the lipid bilayer and generate stable ion currents under a bias voltage. Based on the stable ion transport capabilities of TMEM120A and TMEM120B, as well as the feature of TMEM120A binding with COA-SH, we developed these two proteins into a single-molecule sensor for detecting COA-SH and structurally similar molecules. We found that both COA-SH and ATP can reversibly bind to single TMEM120A and TMEM120B proteins embedded in the lipid bilayer and temporarily block ion currents during the binding process. By analyzing the current blocking signal, COA-SH and ATP can be identified at the single-molecule level. In conclusion, our work has provided two single-molecule biosensors for detecting COA-SH and ATP, offering insights for exploring and developing bio-inspired small molecule sensors.