A simple, novel, and efficient electrochemical sensor was developed for the quantification of Olanzapine (OLA) in its pure form, pharmaceutical formulations, serum, and urine samples. The sensor was based on the modification of a glassy carbon electrode (GCE) with nickel cobaltite/carbon xerogel nanocomposite (NiCo2O4/CX). FESEM, X-ray diffraction (XRD), and N2 adsorption/desorption isotherms were used to characterize the prepared nanocomposite. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to investigate the electrochemical performance of the modified electrode toward electrocatalytic oxidation of OLA. The data obtained showed that the anodic peak of OLA at NiCo2O4/CX/GCE remarkably increased in comparison with bare GCE. The influence of different parameters including nanocomposite amount, accumulation time, scan rate, and pH was studied. Under optimized conditions, the lower limit of detection (LOD) for the DPV oxidation peak current was 4.0 nM, with a linear range of 0.04 µM–212.4 µM. The modified GCE showed good selectivity, excellent repeatability and reproducibility, and high sensitivity. Additionally, NiCo2O4/CX/GCE-based OLA sensor has been successfully utilized for OLA assay in its pharmaceutical formulations, human urine, and serum samples. The developed OLA sensor exhibited good recovery results that agreed with the standard method.