Fast and accurate determination of biological compounds in human fluids plays a crucial role in clinical analysis. Low levels and high levels of ceruloplasmin (Cp) in serum cause several diseases such as Wilson’s disease, therefore, its accurate determination in human fluids is important. A selective and sensitive electrochemical biosensor was developed for Cp detection using a Cp-specific recognition aptamer. The proposed aptasensor was based on a glassy carbon electrode modified with diazonium-functional multiwall carbon nanotubes. The aptamer was linked to the electrode surface using electrochemical approach, followed by chemical immobilization of aminated-aptamer. All fabrication steps were accompanied by changes to the electrochemical parameters. The binding of Cp to aptamer was monitored by cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The effective parameters were optimized. The calibration curve for Cp concentration was linear at 0.02–3.0 ng mL−1 and 3.0–80.0 ng mL−1 with a detection limit (signal-to-noise ratio of 3) of 3.7 pg mL−1. The fabricated aptasensor can serve as a powerful sensor for rapid diagnosis of Cp in the human serum sample and shows a great potential for practical bioapplication.