Electrochemical biosensor is an effective tool for pharmaceutical analysis due to its simplicity, specificity, sensitivity, fast, cost-effective and repetitive measurements with miniaturized and portable devices. The paper illustrates the detail methodology for development of an amperometric biosensor based on polyaniline-gold nanocomposite film modified horseradish peroxidase for anticancer drug gemcitabine in bulk and in parenteral formulation. Scanning Electron Microscopy, Cyclic Voltammetry, Fourier Transform Infra Red Spectroscopy and Electrochemical Impedance Spectroscopic studies of the electrodes and after immobilizing of HRP shows the successful formation of a selectivity of the electrode. The proposed polyaniline-gold nano-composite based biosensor allow quantitation over the range 0.10 to 1.10 ngmL −1 with detection limit of 0.031 ngmL −1 , biosensor sensitivity of 2.934 µAng mL -1 has distinct advantages over other existing methods. Precision and accuracy were also checked and were within the limits. The procedure has been applied to the assay of the drug in dosage form with mean percentage recoveries of 99.00±0.08%. The suggested biosensor method can be successfully applied to the detection and determination of anticancer drug gemcitabine in different drug formulations.