Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatically improved the life expectancy of patients with NSCLC, concerns about TKI-induced cardiotoxicities have increased. AC0010, a novel third-generation TKI, was developed to overcome drug resistance induced by EGFR-T790M mutation. However, the cardiotoxicity of AC0010 remains unclear. To evaluate the efficacy and cardiotoxicity of AC0010, we designed a novel multifunctional biosensor by integrating microelectrodes (MEs) and interdigital electrodes (IDEs) to comprehensively evaluate cell viability, electrophysiological activity, and morphological changes (beating of cardiomyocytes). The multifunctional biosensor can monitor AC0010-induced NSCLC inhibition and cardiotoxicity in a quantitative, label-free, noninvasive, and real-time manner. AC0010 was found to significantly inhibit NCI-H1975 (EGFR-L858R/T790M mutation), while weak inhibition was found for A549 (wild-type EGFR). Negligible inhibition was found in the viabilities of HFF-1 (normal fibroblasts) and cardiomyocytes. With the multifunctional biosensor, we found that 10 μM AC0010 significantly affected the extracellular field potential (EFP) and mechanical beating of cardiomyocytes. The amplitude of EFP continuously decreased after AC0010 treatment, while the interval decreased first and then increased. We analyzed the change in the systole time (ST) and diastole time (DT) within a beating interval and found that the DT and DT/beating interval rate decreased within 1 h after AC0010 treatment. This result probably indicated that the relaxation of cardiomyocytes was insufficient, which may further aggravate the dysfunction. Here, we found that AC0010 significantly inhibited EGFR-mutant NSCLC cells and impaired cardiomyocyte function at low concentrations (10 μM). This is the first study in which the risk of AC0010-induced cardiotoxicity was evaluated. In addition, novel multifunctional biosensors can comprehensively evaluate the antitumor efficacy and cardiotoxicity of drugs and candidate compounds.