In this study, we designed a simple, rapid, sensitive and selective surface plasmon resonance (SPR) sensor for detection of L-phenylalaine by utilizing molecular imprinting technology. l-phenylalanine imprinted and non-imprinted poly(2-hydroxyethyl methacrylate-methacryloyl-l-phenylalanine) polymeric films were synthesized onto SPR chip surfaces using ultraviolet polymerization. l-phenyalanine imprinted and non-imprinted SPR sensors were characterized by using contact angle, atomic force microscopy and ellipsometry. After characterization studies, kinetic studies were carried out in the concentration range of 5.0-400.0 mu M. The limit of detection and quantification were obtained as 0.0085 and 0.0285 mu M, respectively. The response time for the test including equilibration, adsorption and desorption was approximately 9 min. The selectivity studies of the l-phenylalanine imprinted SPR sensor was performed in the presence of d-phenylalanine and l-tryptophan. Validation studies were carried out via enzyme-linked immunosorbent analysis technique in order to demonstrate the applicability and superiority of the l-phenylalanine imprinted SPR sensor.