For selective and sensitive determination of L-phenylalanine (L-Phe) a novel and reliable electrochemical sensor has been developed based on molecular imprinting technique. Thiophen-3-carbonyl tryptophan (TP3C-Trp) has been synthesized and used as a novel monomer. L-Phe was used as template molecule and imprinted in presence of thiophen and thiophen-3-carbonyl tryptophan (TP3C-Trp), used as functional monomers. As a comparison a non-modified polymer surface was also prepared under same circumstances without template molecule, L-Phe. The characterization studies of both non-imprinted (NIP) and L-phenylalanine imprinted (MIP) electrochemical sensor surfaces were made by using atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), ellipsometry and contact angle measurements. The electrochemical characterization analysis were made via square wave and cyclic voltammetry. The linearity range and the detection limit of the developed sensor were obtained as 1.0 x 10(-8)-1.0 x 10(-7) M and 1.0 x 10(-9) M, respectively.