Glutathione-capped copper sulfide (CuxSy) nanoparticles with two different average sizes were successfully achieved by using a simple reduction process that involves only changing the reaction temperature. Temperature-induced changes in the size of CuxSy nanoparticles resulted in particles with different optical, morphological, and electrochemical properties. The dependence of electrochemical sensing properties on the sizes of CuxSy nanoparticles was studied by using voltammetric and amperometric techniques. The spherical CuxSy nanoparticles with the average particle size of 25 ± 0.6 nm were found to be highly conductive as compared to CuxSy nanoparticles with the average particle size of 4.5 ± 0.2 nm. The spherical CuxSy nanoparticles exhibited a low bandgap energy (Eg) of 1.87 eV, resulting in superior electrochemical properties and improved electron transfer during glucose detection. The sensor showed a very good electrocatalytic activity toward glucose molecules in the presence of interference species such as uric acid (UA), ascorbic acid (AA), fructose, sodium chloride, and sucrose. These species are often present in low concentrations in the blood. The sensor demonstrated an excellent dynamic linear range between 0.2 to 16 mM, detection limit of 0.2 mM, and sensitivity of 0.013 mA/mM. The applicability of the developed sensor for real field determination of glucose was demonstrated by use of spiked blood samples, which confirmed that the developed sensor had great potential for real analysis of blood glucose levels.