This study aimed to develop a molecularly imprinted polymer (MIP) sensor using electropolymerization of thiophene acetic acid monomer around template molecules, sulfaguanidine (SGN) and sulfamerazine (SMR), for selective and sensitive detection of both antibiotics. Au nanoparticles were then deposited on the modified electrode surface, and SGN and SMR were extracted from the resulting layer. Surface characterization, changes in the oxidation peak current of both analytes, and investigation of the electrochemical properties of the MIP sensor were examined using scanning electron microscopy, cyclic voltammetry, and differential pulse voltammetry. The developed MIP sensor with Au nanoparticles showed a detection limit of 0.030 µmol L−1 and 0.046 µmol L−1 for SGN and SMR, respectively, with excellent selectivity in the presence of interferents. The sensor was successfully used for SGN and SMR analysis in human fluids, including blood serum and urine, with excellent stability and reproducibility.