Biosensing technologies have demonstrated significant potential in exploring the binding of drugs to receptor tyrosine kinases (RTKs). As a typical transmembrane receptor, there are still several shortcomings in the utilization of the intracellular kinase domain of RTKs, the primary action site of small-molecule inhibitors, resulting in insufficient binding and unclear action sites, which impair the efficiency and accuracy of biosensing. Herein, using epidermal growth factor receptor (EGFR) as an example, we reported a biosensing platform based on cell membrane camouflage technology for evaluating drugs binding to the intracellular kinase domain of EGFR. The azide-functionalized cell membranes modified through glucose metabolism were reverse-coated onto alkyne-functionalized magnetic nanoparticles via bioorthogonal reaction (CMRMNPs), therefore effectively exposing the intracellular kinase domain of EGFR without damage. To construct the biosensing platform, a small-molecule fluorescent probe derived from the gefitinib pharmacophore (GN probe) was further synthesized and incubated with CMRMNPs. This strategy facilitated the efficient localization of the GN probe within the intracellular kinase domain of EGFR. Ultimately, this approach was successfully implemented to evaluate the binding of three inhibitors with EGFR. This study provides a viable strategy for constructing biomimetic biosensors with a defined cell membrane orientation and offers novel insights and methodologies for the study of drug binding with the intracellular kinase regions of RTKs.