Protein phosphorylation plays an important role in many biological processes and might be used as a potential biomarker in clinical diagnoses. We reported the development of a nanomaterial enhanced disposable immunosensor for ultrasensitive detection of phosphorylated p53 at Ser392 (phospho-p53³⁹²) using enzyme functionalization of carbon nanospheres (CNSs) as a signal amplification label and magnetic beads (MB) coupled with screen printed carbon electrodes as electrochemical transducers. In this work, horseradish peroxidase (HRP) and phospho-p53³⁹² detection antibody (Ab₂) were co-linked to CNSs (HRP–CNSs–Ab₂) for signal amplification, and functionalized MB was used as a platform to capture a large amount of primary antibodies (Ab₁). The proposed signal amplification strategy with a sandwich-type immunoreaction significantly enhanced the sensitivity of detection of biomarkers. Under optimal conditions, the immunosensor had a highly linear voltammetric response to the phospho-p53³⁹² concentration in the range of 0.01 to 5 ng mL⁻¹, with a detection limit of 3.3 pg mL⁻¹. The results provided great potential for point-of-care detection of other phosphorylated proteins and clinical applications.