Molecularly imprinted polymer (MIP) for sulfanilamide (SN) sensing is prepared through in-situ electropolymerization of pyrrole (Py) on pencil graphite electrode (PGE). Computational study using density functional theory with B3LYP level is performed to analyze and evaluate the template-monomer geometry. Among the various functional monomers studied pyrrole is found to have the highest binding interaction energy with SN and it is chosen as a functional monomer. Electropolymerization of pyrrole in the presence of SN on PGE is carried out using cyclic voltammetry. Structural morphology of the imprinted polymer is characterized by field emission scanning electron microscopy (FESEM) studies. Quantitative measurements of MIP based SN detection are performed by using cyclic voltammetry and differential pulse voltammetry studies. Several important kinetic parameters influencing the performance of SN sensor such as limit of detection, linear concentration range and sensitivity etc. are determined and analyzed. Under the optimized conditions, MIP based SN sensor proposed in this work has a very low detection limit of 2.0×10-8M (S/N=3) and possesses two linear ranges from 5.0×10-8 to 1.1×10-6M and 1.1×10-6 to 48×10-6M with a sensitivity value of 1.158 and 0.012μA/μM respectively. This particular sensor shows a good selectivity towards SN in presence of potential other structural analogue interferences namely sulfamethaxazole, sulfathiazole and sulfadiazine. Furthermore, the fabricated sensor is successfully applied to quantitatively determine and analyze SN present in spiked human serum and ground water samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.