Glutamate is an important neurotransmitter due to its critical role in physiological and pathological processes. While enzymatic electrochemical sensors can selectively detect glutamate, enzymes cause instability of the sensors, thus necessitating the development of enzyme-free glutamate sensors. In this paper, we developed an ultrahigh sensitive nonenzymatic electrochemical glutamate sensor by synthesizing copper oxide (CuO) nanostructures and physically mixing them with multiwall carbon nanotubes (MWCNTs) onto a screen-printed carbon electrode. We comprehensively investigated the sensing mechanism of glutamate; the optimized sensor showed irreversible oxidation of glutamate involving one electron and one proton, and a linear response from 20 μM to 200 μM at pH 7. The limit of detection and sensitivity of the sensor were about 17.5 μM and 8500 μA·mM−1·cm−2, respectively. The enhanced sensing performance is attributed to the synergetic electrochemical activities of CuO nanostructures and MWCNTs. The sensor detected glutamate in whole blood and urine and had minimal interference with common interferents, suggesting its potential for healthcare applications.