Breast cancer (BC) is one of the most common types of cancer disease worldwide and it accounts for thousands of deaths annually. Lapatinib is among the preferred drugs for the treatment of breast cancer. Possible drug toxicity effects of lapatinib can be controlled by real-time determination of the appropriate dose for a patient at the point of care. In this study, a novel highly sensitive polymeric nanobiosensor for lapatinib is presented. A composite of poly(anilino-co-4-aminobenzoic acid) co-polymer {poly(ANI-co-4-ABA)} and coffee extract-based green-synthesized indium nanoparticles (InNPs) was used to develop the sensor platform on a screen-printed carbon electrode (SPCE), i.e., SPCE||poly(ANI-co-4-ABA-InNPs). Cytochrome P450-3A4 (CYP3A4) enzyme and polyethylene glycol (PEG) were incorporated on the modified platform to produce the SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG lapatinib nanobiosensor. Experiments for the determination of the electrochemical response characteristics of the nanobiosensor were performed with cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The nanobiosensor calibration for 0–100 ng/mL lapatinib was linear and gave limit of detection (LOD) values of 13.21 ng/mL lapatinib and 18.6 ng/mL lapatinib in physiological buffer and human serum, respectively. The LOD values are much lower than the peak plasma concentration (Cmax) of lapatinib (2.43 µg/mL), which is attained 4 h after the administration of a daily dose of 1250 mg lapatinib. The electrochemical nanobiosensor also exhibited excellent anti-interference performance and stability.