In the present work, an innovative electrochemical sensor was fabricated based on poly toluidine blue modified glassy carbon electrode (PTB-GCE). So, PTB-GCE was used for the detection and determination of doxorubicin hydrochloride (DOX) in cell lysate, and whole human plasma samples. PTB could enhance the rate of electrochemical reaction for the electro oxidation and detection of DOX in real samples. Cyclic voltammetry (CV) technique was used for the electro polymerization of toluidine blue on the surface of GCE with the applied potential ranging from -0.6 to 0.2 V. The sensor construction steps were approved by field emission scanning electron microscopy (FE-SEM), Energy dispersive X-ray spectroscopy (EDX) and electrochemical methods. Also, CV results indicated that the DOX is oxidized via two electrons and two protons process at the optimum pH of 6.5 using PTB modified GCE. Under optimized conditions, differential pulse voltammetry (DPV) technique response exhibited linear relationship between the oxidative peak current and concentration of DOX in the range of 17 nM - 8.6 μM with low limit of quantification (LLOQ) of 17 nM for untreated and treated human plasma samples. Also, determination of DOX in MDA-MB-231 and 4T1cell lysates were performed based on its direct electrochemical oxidation on PTB-GCE. Finally, analytical validation of DOX in human bio-fluids using FDA guideline were done successfully. Results suggested that the proposed electrochemical sensor can be used to the sensitive and selective determination of DOX in biological samples. The interaction results of DOX with cancer cells indicate the developed probe can easily detect candidate drug in cancer cells with high accuracy. To the best of our knowledge this is the first report of the determination of DOX based on the direct electrochemical oxidation on PTB-GCE and determination in MDA-MB-231 and 4T1 cell lysates. It is anticipated that this research open new horizons on the design of new class of electrochemical sensors for determination drugs, and therapeutic drug monitoring (TDM) in human bio-fluids.