This work describes the implementation of a low-cost, robust, and sensitive sensor for the efficiency and rapid electrochemical detection of traces of methylene blue (MB) in contaminated aqueous media. An organohydroxyapatite (OHAP) hybrid material was prepared by cross-linking grafting β-cyclodextrin (β-CD) with citric acid (CA) at the surface of an impregnated natural hydroxyapatite (HAPI). The structural characterization of HAPI and OHAP materials was performed using SEM–EDX. Thin films of different materials were deposited on the surface of a glassy carbon electrode (GCE); their surface charge and permeability were evaluated by multisweep cyclic voltammetry using [Fe(CN)6]3−/4− and [Ru(NH3)6]3+/2+ as redox probe systems. The electrochemical behavior of MB on the GCE modified by a film of HAPI or OHAP was also studied by cyclic voltammetry. As with [Ru(NH3)6]3+ ions, the cyclic voltammograms obtained showed that MB molecules are quantitatively and progressively accumulated on OHAP thin film. Then, the GCE/OHAP electrode was exploited to build a sensitive sensor for the detection of MB. In comparison with the bare GCE, the GCE/OHAP exhibited more sensitive electrochemical response toward MB. Under optimized conditions, a calibration curve was obtained in the large concentration range from 5.0 × 10–8 to 400.0 × 10–8 mol L−1, leading to a limit of detection of 2.15 × 10–9 mol L−1 (S/N = 3). The developed sensor GCE/OHAP was successfully applied to the electroanalytical sensitive quantification of MB in environmental water samples, such as spring water, river water, and well water.