The development of nano biocomposite film (f-MWCNTs-Au-GOx) for L-cysteine (LC) detection is proposed byusing glassycarbon electrode (GCE). The proposed nano biocomposite film has been fabricated on ITO for scanning electron microscopy(SEM) and atomic force microscopy(AFM) analysis. Next, the f-MWCNTs-Au-GOx nano biocomposite film modified GCE’s surface was examined byscanning electrochemical microscopy(SECM). The proposed nano biocomposite film has been successfullyapplied for the detection of LC using cyclic voltammetry(CV) and amperometry. Thef-MWCNTs-Au-GOx film modified GCE exhibited a linear response for LC detection in the lower and higher concentrations ranges of 2 to 42×10−6, 0.1 to 1.08×10−3 mol L−1. Also, the proposed nano biocomposite film possesses high sensitivityand good repeatabilityf or LC detection.