Herein, a surface plasmon resonance (SPR)-based nanosensor was fabricated using molecular imprinting technique for the selective and sensitive detection of amitrole, a commonly used highly toxic pesticide. For this purpose, polymeric nanofilms involving N-methacryloyl-l-tryptophan methyl ester and ethylene glycol dimethacrylate which are functional monomer and cross-linker, respectively, were prepared on the SPR chips surface via the ultravoilet polymerization method. The nanofilms were characterized by several techniques such as ellipsometry, Fourier transform infrared-attenuated total reflection, atomic force microscope and contact angle measurements. The kinetic analyses for amitrole detection were examined through amitrole-imprinted and non-imprinted SPR sensor chips. The developed imprinted sensor showed high selectivity to the amitrole molecule than similar molecules and a good linear relationship for the 0.06-11.90 nM concentration range with a low limit of detection value of 0.037 nM. The high imprinting efficiency (I.F: 62.38) of the amitrole-imprinted SPR sensor was determined by comparing it with the non-imprinted SPR sensor. The most suitable model of this sensor is the Langmuir adsorption model. To statistically assess the reusability of the sensor, intraday experiments were tested three times with five replicates. The relative standard deviation% value less than <1.5 indicates high reproducibility for both sensor production and reproducibility of the method. The prepared sensor was also applied efficiently for the selective detection of the amitrole in spiked samples prepared from vegetables to evaluate the matrix effect. As a result, amitrole-imprinted SPR sensors have been estimated to be highly selective, fast responsive, easy to use, reusable and sensitive in detecting amitrole in both natural source and aqueous solutions. Label-free amitrole detection was performed by an aminoacid-based SPR sensor fabricated without the need for complex coupling processes.