The global surge in chronic hyperglycemia, the autoimmune disease resulting from diabetes mellitus, has attracted worldwide scientists to develop novel methods for assaying blood glucose levels in the human body. In this work, we reported a new sensitive chromogenic reagent-based single beam spectrophotometric assay of glucose. This method is based on the catalytic oxidation of D-glucose to D-gluconolactone and H 2 O 2 by glucose oxidase (GOD), later by the oxidative coupling of quinone with imine radical to give purple-colored quinone-imine chromogenic species with λ max = 540 nm. The linearity of glucose lies in the range of 57.81-1850.2 and 14.45-925.1 µmol L -1 by rate and fixed time method. The Michaelis-Menten constant attained by the Lineweaver-Burk plot at the optimal concentration of the reagents was 526.31 µM with V max of 0.2659 EU min -1 and catalytic constant of 1.3295 min -1 . Intra-and inter-day precision ranged between 0.73%-1.4% and 1.76%-2.4%, respectively. The standard addition method for the recovery of glucose fluctuated between 97.78% and 104.35%, registering minimal common interference species present in the blood samples. The results showed a good accuracy level (90%-102%), and the calculated limit of detection (LOD) and limit of quantification (LOQ) were as low as 2.17 and 8.56 µM. The proposed work has a good correlation with the standard enzyme kit method. Hence, the proposed system can be convenient for the determination of glucose in biological samples.