Calretinin (CAL) is calcium binding protein, and its levels in blood and cerebrospinal fluids are increased, since its expression is increased various cancer types. A novel biosensor system fabricated by immobilization of a specific antibody to CAL, anti-Calretinin (anti-CAL), onto a gold electrode surface via an effective covalent binding method using mercaptohexanol, epichlorohydrin, and ethanolamine was reported for the sensitive, selective, and accurate analysis of CAL. The proposed biosensor showed a linear calibration range between 1 ng/mL and 5 ng/mL. LOD and LOQ values were determined as 0.11 ng/mL and 0.38 ng/mL, respectively. The standard deviation related to the reproducibility of the new biosensor system was calculated as 3.95%. Lastly, in order to state the applicability of the biosensor to early diagnosis of CAL in practice, artificial serum samples spiked with CAL have been analyzed by the proposed biosensor.