A chiral electrochemical sensor was developed via an electro-generated molecularly imprinted polymer-based ultrathin film using L-ascorbic acid as a template and its biological proficiency was analyzed. The performance of the chiral selective sensor in serum was evaluated by conducting a comparative study of electrochemical response of L-ascorbic acid imprinted polyaniline ferrocene sulfonic acid pencil graphite electrode in aqueous and serum samples employing cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The sensor shows similar electrochemical response in aqueous and serum solutions of L-ascorbic acid of equal concentrations even at considerably high serum proportion in solutions. This shows its excellent ability to counter multifold interference in extraordinary complex biological medium (serum) and hence validates its worth in clinical applications. Surface morphologies are characterized through scanning electron microscopy of the chiral sensor (before and after de-doping of L-ascorbic acid) and compared with bare and non-imprinted electrode. Electrochemical impedance spectroscopy technique is used for the molecularly imprinted electrodes to quantitatively measure L-ascorbic acid with a detection limit of 1 mu M. Finally, the proposed sensor offers excellent prospects in biomedical fields for simple, fast and cost effective chiral quantification of ascorbic acid in serum samples.